Microcarrier perfusion culturing methods and uses thereof

ABSTRACT

Provided herein are methods of culturing a mammalian cell and various methods that utilize these culturing methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/769,783, filed Aug. 21, 2015, which is a 371 U.S. National StageApplication of PCT Application No. PCT/US2014/017785, filed Feb. 21,2014, which claims priority to U.S. Provisional Patent Application Ser.No. 61/768,215, filed Feb. 22, 2013, the entire contents of which areherein incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of molecular biology, cell cultureprocess development, and the manufacture of recombinant proteins.

BACKGROUND

Mammalian cells containing a nucleic acid that encodes a recombinantprotein are often used to produce therapeutically or commerciallyimportant proteins. Although several high throughput (HT) cell culturesystems have been used within the biotechnology industry for fed-batchprocesses for years, no HT model for a perfusion-based cell cultureusing shake tubes and microcarriers is known to exist.

Previous methods of mammalian tissue culture using shake tubes for feedbatch cultures or perfusion cultures can produce recombinant proteins athigh cell density; however, previous methods of culturing cells using ashake tube and microcarriers have been unsuccessful because of the shearstress inflicted on the mammalian cells by the circulatingmicrocarriers, which results in a decrease in cell growth. In addition,it is difficult to harvest a recombinantly produced protein from a shaketube culture containing microcarriers.

SUMMARY

The present invention is based, at least in part, on the discovery thatculturing a mammalian cell in the specific manner described herein(including the use of a shake tube and a plurality of microcarriers)results in actively growing mammalian cell cultures that effectivelyreplicate the recombinant protein production achieved in a larger scalecontinuous-perfusion bioreactor containing microcarriers. Thus, thepresent specification includes methods of culturing a mammalian cellthat include: providing a shake tube containing a mammalian celldisposed in a first liquid culture medium, where the first liquidculture medium occupies, e.g., about 10% to about 30% of the volume ofthe shake tube, and contains a plurality of microcarriers at aconcentration of about 1.0 g/L to about 15.0 g/L; incubating the shaketube for a period of time at about 32° C. to about 39° C. and with arotary agitation of about 120 revolutions per minute (RPM) to about 240RPM (e.g., about 120 RPM to about 160 RPM); and after about the first 48to 96 hours of the period of time, continuously or periodically removinga first volume of the first liquid culture medium and adding to thefirst liquid culture medium a second volume of a second liquid culturemedium, where the first and second volumes are about equal. Alsoprovided are various methods that utilize these culturing methods.

Provided herein are methods of culturing a mammalian cell. These methodsinclude providing a shake tube containing a mammalian cell disposed in afirst liquid culture medium, where the first liquid culture mediumoccupies about 10% to about 30% of the volume of the shake tube andcontains a plurality of microcarriers at a concentration of about 1.0g/L to about 15.0 g/L; incubating the shake tube for a period of time atabout 32° C. to about 39° C. and with a rotary agitation of about 120revolutions per minute (RPM) to about 240 RPM; and after about the first48 to 96 hours of the period of time, continuously or periodicallyremoving a first volume of the first liquid culture medium and adding tothe first liquid culture medium a second volume of a second liquidculture medium, where the first and second volumes are about equal. Insome embodiments of these methods, the first volume of the first liquidculture medium is substantially free of the microcarriers. In someembodiments of these methods, at the beginning of the period of time,the first liquid culture medium contains 0.1×10⁶ cells/mL to 0.5×10⁶cells/mL. In some embodiments of any of these methods, the mammaliancell is a Chinese hamster ovary (CHO) cell. In some embodiments of anyof these methods, the CHO cell contains a nucleic acid encoding arecombinant protein. In some embodiments of any of these methods, therecombinant protein is an immunoglobulin, an enzyme, a growth factor, aprotein fragment, or an engineered protein. In some embodiments of anyof these methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium is performed simultaneously. In some embodiments of anyof these methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium is performed continuously. In some embodiments of any ofthese methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium is performed periodically. In some embodiments of any ofthese methods, the first volume of the first liquid culture mediumremoved and the second volume of the second liquid culture medium addedare increased over time. In some embodiments of any of these methods,the first liquid culture medium is the same as the second liquid culturemedium. In some embodiments of any of these methods, the first liquidculture medium is different from the second liquid culture medium. Insome embodiments of any of these methods, the shake tube has a volume ofbetween about 10 mL to about 100 mL. In some embodiments of any of thesemethods, the mammalian cell is suspended in about 2 mL to about 20 mL ofthe first liquid culture medium. In some embodiments of any of thesemethods, the first liquid culture medium and/or second liquid culturemedium is selected from the group consisting of: a chemically-definedliquid culture medium, a serum-free liquid culture medium, aserum-containing liquid culture medium, an animal-derived component freeliquid culture medium, and a protein-free medium. In some embodiments ofany of these methods, after about the first 48 to 96 hours of the periodof time, in each 24-hour period, the first volume of the first liquidculture medium removed and the second volume of the second liquidculture medium added is about 30% to about 95% of the volume of thefirst liquid culture medium. In some embodiments of any of thesemethods, the agitation is ceased for a period of time of at least 30seconds prior to removing the first volume of the first liquid culturemedium. In some embodiments of any of these methods, the plurality ofmicrocarriers has a mean diameter of between about 200 μm to about 800μm. In some embodiments of any of these methods, the plurality ofmicrocarriers contains one or more pores. In some embodiments of any ofthese methods, the one or more pores has a mean diameter of about 25 μmto about 35 μm. In some embodiments of any of these methods, the shaketube is incubated at a reactor angle of about 25 degrees to about 90degrees from horizontal.

Also provided are are methods of culturing a mammalian cell thatinclude: (a) providing a shake tube containing a mammalian cell disposedin a first liquid culture medium that occupies about 10% to about 30% ofthe volume of the shake tube and contains a plurality of microcarriersin a concentration of about 1.0 g/L to about 15.0 g/L; (b) incubatingthe shake tube for a first time period at about 35° C. to about 39° C.with a rotary agitation of about 120 revolutions per minute (RPM) toabout 240 RPM, and after about the first 48 to 96 hours of the firstperiod of time, in each subsequent 24-hour period, (i) continuously orperiodically removing a first volume of the first liquid culture mediumthat is substantially free of microcarriers from the shake tube, wherethe first volume is about 10% to about 95% of the volume of the firstliquid culture medium; and (ii) adding to the shake tube a second volumeof a second liquid culture medium, where the first and second volumesare about equal; (c) incubating the shake tube after the cellconcentration reaches about target cell density for a second time periodof about 2 days to about 7 days, at about 32° C. to about 39° C. withthe rotary agitation, and in each 24-hour period, performing steps(b)(i) and (b)(ii),where the first and second liquid culture media usedin step (b) are of a substantially different type from those used instep (c); and (d) incubating the shake tube for a third time periodgreater than 2 days, at about 35° C. to about 39° C. with the rotaryagitation, and in each 24-hour period, performing steps (b)(i) and(b)(ii), where the first and second liquid culture media used in step(c) are of the same type as those used in step (d).

In some embodiments of any of these methods, at the beginning of thefirst period of time, the first liquid culture medium contains 0.1×10⁶cells/mL to 0.5×10⁶ cells/mL. In some embodiments of any of thesemethods, the mammalian cell is a Chinese hamster ovary (CHO) cell. Insome embodiments of any of these methods, the CHO cell contains anucleic acid encoding a recombinant protein. In some embodiments of anyof these methods, the recombinant protein is a secreted immunoglobulin,a secreted enzyme, a secreted growth factor, a secreted proteinfragment, or a secreted engineered protein. In some embodiments of anyof these methods, the removing of the first volume of the first liquidculture medium, and the adding of the second volume of the second liquidculture medium in one or more of the first time period, the second timeperiod, and the third time period is performed simultaneously. In someembodiments of any of these methods, the removing of the first volume ofthe first liquid culture medium, and the adding of the second volume ofthe second liquid culture medium in one or more of the first timeperiod, the second time period, and the third time period is performedcontinuously. In some embodiments of any of these methods, the removingof the first volume of the first liquid culture medium, and the addingof the second volume of the second liquid culture medium in one of moreof the first time period, the second time period, and the third timeperiod is performed periodically. In some embodiments of any of thesemethods, the shake tube has a volume of between about 10 mL to about 100mL. In some embodiments of any of these methods, the volume of the firstliquid culture medium is about 2 mL to about 20 mL. In some embodimentsof any of these methods, the first liquid culture medium and secondliquid culture medium used in the first time period is serum-containingliquid culture medium or an animal-derived component-containing liquidculture medium, and the first liquid culture medium and the secondliquid culture medium used in the second time period and the third timeperiod is a serum-free liquid culture medium, an animal-derivedcomponent-free liquid culture medium, or a protein- free medium. In someembodiments of any of these methods, the agitation is ceased for atleast 30 seconds prior to removing the first volume of the first liquidculture medium from the shake tube during one or more of the first timeperiod, the second time period, and the third time period. In someembodiments of any of these methods, the plurality of microcarriers hasa mean diameter of between about 200 μm to about 800 μm. In someembodiments of any of these methods, the plurality of microcarrierscontains one or more pores. In some embodiments of any of these methods,the one or more pores has a mean diameter of about 25 μm to about 35 μm.In some embodiments of any of these methods, the first volume of thefirst liquid culture medium removed during the third period of timecontains a substantial number of microcarriers. In some embodiments ofany of these methods, the first volume of the first liquid culturemedium removed during the third period of time is substantially free ofmicrocarriers. In some embodiments of any of these methods, the firstvolume of the first liquid culture medium removed and the second volumeof the second liquid culture medium added in one or more of the firsttime period, the second time period, and the third time period is about70% of the volume of the first liquid culture medium. In someembodiments of any of these methods, the shake tube is incubated in oneor more of (b), (c), and (d) at a reactor angle of about 25 degrees toabout 90 degrees from horizontal.

Also provided are methods of producing a recombinant protein. Thesemethods include: providing a shake tube containing a mammalian cellcontaining a nucleic acid encoding a recombinant protein disposed in afirst liquid culture medium, where the first liquid culture mediumoccupies about 10% to about 30% of the volume of the shake tube andcontains a plurality of microcarriers at a concentration of about 1.0g/L to about 15.0 g/L; incubating the shake tube for a period of time atabout 32° C. to about 39° C. and with a rotary agitation of about 120revolutions per minute (RPM) to about 240 RPM; and after about the first48 to 96 hours of the period of time, continuously or periodicallyremoving a first volume of the first liquid culture medium and adding tothe first liquid culture medium a second volume of a second liquidculture medium, where the first and second volumes are about equal; andrecovering the recombinant protein from the mammalian cell or from thefirst and/or second liquid culture medium. In some embodiments of any ofthese methods, the recombinant protein is recovered from the mammaliancell. In some embodiments of any of these methods, the recombinantprotein is recovered from the first and/or second liquid culture medium.In some embodiments of any of these methods, the first volume of thefirst liquid culture medium removed is substantially free ofmicrocarriers. In some embodiments of any of these methods, at thebeginning of the period of time, the first liquid culture mediumcontains 0.1×10⁶ cells/mL to 0.5×10⁶ cells/mL. In some embodiments ofany of these methods, the mammalian cell is a Chinese hamster ovary(CHO) cell. In some embodiments of any of these methods, the recombinantprotein is an immunoglobulin, an enzyme, a growth factor, a proteinfragment, or an engineered protein. In some embodiments of any of thesemethods, the recombinant protein is secreted into the first and/orsecond liquid culture medium. In some embodiments of any of thesemethods, the removing of the first volume of the first liquid culturemedium and the adding of the second volume of the second liquid culturemedium is performed simultaneously. In some embodiments of any of thesemethods, the removing of the first volume of the first liquid culturemedium and the adding of the second volume of the second liquid culturemedium is performed continuously. In some embodiments of any of thesemethods, the removing of the first volume of the first liquid culturemedium and the adding of the second volume of the second liquid culturemedium is performed periodically. In some embodiments of any of thesemethods, the first volume of the first liquid culture medium removed andthe second volume of the second liquid culture medium added areincreased over time. In some embodiments of any of these methods, thefirst liquid culture medium is the same as the second liquid culturemedium. In some embodiments of any of these methods, the first liquidculture medium is different from the second liquid culture medium. Insome embodiments of any of these methods, the shake tube has a volume ofbetween about 10 mL to about 100 mL. In some embodiments of any of thesemethods, the mammalian cell is suspended in about 2 mL to about 20 mL ofthe first liquid culture medium. In some embodiments of any of thesemethods, the first liquid culture medium and/or second liquid culturemedium is selected from the group of: a chemically-defined liquidculture medium, a serum-free liquid culture medium, a serum-containingliquid culture medium, an animal-derived component-free liquid culturemedium, and a protein-free medium. In some embodiments of any of thesemethods, after about the first 48 to 96 hours of the period of time, ineach 24-hour period, the first volume of the first liquid culture mediumremoved and the second volume of the second liquid culture medium addedis about 30% to about 95% of the volume of the first liquid culturemedium. In some embodiments of any of these methods, the agitation isceased for a period of time of at least 30 seconds prior to removing thefirst volume of the first liquid culture medium. In some embodiments ofany of these methods, the plurality of microcarriers has a mean diameterof between about 200 μm to about 800 In some embodiments of any of thesemethods, the plurality of microcarriers contains one or more pores. Insome embodiments of any of these methods, the one or more pores has amean diameter of about 25 μm to about 35 In some embodiments of any ofthese methods, the shake tube is incubated at a reactor angle of about25 degrees to about 90 degrees from horizontal.

Also provided are methods of producing a recombinant protein thatinclude: (a) providing a shake tube containing a mammalian cellcontaining a nucleic acid encoding a recombinant protein disposed in afirst liquid culture medium, where the first liquid culture mediumoccupies about 10% to about 30% of the volume of the shake tube andcontains a plurality of microcarriers in a concentration of about 1.0g/L to about 15.0 g/L; (b) incubating the shake tube for a first timeperiod at about 35° C. to about 39° C. with a rotary agitation of about120 revolutions per minute (RPM) to about 240 RPM, and after about thefirst 48 hours to 96 hours of the first period of time, in eachsubsequent 24-hour period, (i) continuously or periodically removing afirst volume of the first liquid culture medium that is substantiallyfree of microcarriers from the shake tube, where the first volume isabout 10% to about 95% of the volume of the first liquid culture medium;and (ii) adding to the shake tube a second volume of a second liquidculture medium, where the first and second volumes are about equal; (c)incubating the shake tube after the cell concentration reaches abouttarget cell density for a second time period of about 2 days to about 7days, at about 32° C. to about 39° C. with the rotary agitation, and ineach 24-hour period, performing steps (b)(i) and (b)(ii), where thefirst and second liquid culture media used in step (b) are of asubstantially different type from those used in step (c); (d) incubatingthe shake tube for a third time period greater than 2 days, at about 35°C. to about 39° C. with the rotary agitation, and in each 24-hourperiod, performing steps (b)(i) and (b)(ii), where the first and secondliquid culture media used in step (c) are of the same type as those usedin step (d); and (e) recovering the recombinant protein from themammalian cell or the first and/or second liquid culture medium usedduring the first, second, and/or third period of time. In someembodiments of any of these methods, the recombinant protein isrecovered from the mammalian cell. In some embodiments of any of thesemethods, the recombinant protein is recovered from the first and/orsecond liquid culture medium used during one or more of the first,second, and third period of time. In some embodiments of any of thesemethods, at the beginning of the first period of time, the first liquidculture medium contains 0.1×10⁶ cells/mL to 0.5×10⁶ cells/mL. In someembodiments of any of these methods, the mammalian cell is a Chinesehamster ovary (CHO) cell. In some embodiments of any of these methods,the recombinant protein is an immunoglobulin, an enzyme, a growthfactor, a protein fragment, or an engineered protein. In someembodiments of any of these methods, the recombinant protein is secretedinto the first and/or second liquid culture medium used during one ormore of the first period of time, the second period of time, and thethird period of time. In some embodiments of any of these methods, theremoving of the first volume of the first liquid culture medium and theadding of the second volume of the second liquid culture medium in oneor more of the first time period, the second time period, and the thirdtime period is performed simultaneously. In some embodiments of any ofthese methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium in one or more of the first time period, the second timeperiod, and the third time period is performed continuously. In someembodiments of any of these methods, the removing of the first volume ofthe first liquid culture medium and the adding of the second volume ofthe second liquid culture medium in one of more of the first timeperiod, the second time period, and the third time period is performedperiodically. In some embodiments of any of these methods, the shaketube has a volume of between about 10 mL to about 100 mL. In someembodiments of any of these methods, the volume of the first liquidculture medium is about 2 mL to about 20 mL. In some embodiments of anyof these methods, the first liquid culture medium and second liquidculture medium used in the first time period is serum-containing liquidculture medium or an animal-derived component-containing liquid culturemedium, and the first liquid culture medium and second liquid culturemedium used in the second time period and the third time period is aserum-free liquid culture medium, an animal-derived component freeliquid culture medium, or a protein-free medium. In some embodiments ofany of these methods, the agitation is ceased for at least 30 secondsprior to removing the first volume of the first liquid culture medium,from the shake tube during one or more of the first time period, thesecond time period, and the third time period. In some embodiments ofany of these methods, the plurality of microcarriers has a mean diameterof between about 200 μm to about 800 μm. In some embodiments of any ofthese methods, the plurality of microcarrier contains one or more pores.In some embodiments of any of these methods, the one or more pores has amean diameter of about 25 μm to about 35 μm. In some embodiments of anyof these methods, the first volume of the liquid culture medium removedduring the third period of time contains a substantial number ofmicrocarriers. In some embodiments of any of these methods, the firstvolume of the liquid culture medium removed during the third period oftime is substantially free of microcarriers. In some embodiments of anyof these methods, the first volume of the first liquid culture mediumremoved and the second volume of the second liquid culture medium addedin one or more of the first time period, the second time period, and thethird time period is about 70% of the volume of the first liquid culturemedium. In some embodiments of any of these methods, the shake tube isincubated in one or more of (b), (c), and (d) at a reactor angle ofabout 25 degrees to about 90 degrees from horizontal.

Also provided are methods for testing a manufacturing process for makinga recombinant protein. These methods include: providing a shake tubecontaining a mammalian cell containing a nucleic acid encoding arecombinant protein disposed in a first liquid culture medium, where thefirst liquid culture medium occupies about 10% to about 30% of thevolume of the shake tube and contains a plurality of microcarriers at aconcentration of about 1.0 g/L to about 15.0 g/L; incubating the shaketube for a period of time at about 32° C. to about 39° C. and with arotary agitation of about 120 revolutions per minute (RPM) to about 240RPM; after about the first 48 to 96 hours of the period of time,continuously or periodically removing a first volume of the first liquidculture medium and adding to the first liquid culture medium a secondvolume of a second liquid culture medium, where the first and secondvolumes are about equal; detecting the recombinant protein in the cellor in the first and/or second culture medium; and comparing the amountof recombinant protein present in the cell or in the first and/or secondculture medium to a reference level of recombinant protein. In someembodiments of any of these methods, the first volume of the firstliquid culture medium is substantially free of mammalian cells. In someembodiments of any of these methods, the reference level of recombinantprotein is a level of recombinant protein produced using a differentculturing method. In some embodiments of any of these methods, thedifferent culturing method utilizes a different first or second liquidculture medium, a different mammalian cell, a different temperature, adifferent level of agitation, a different shake tube, or a differentmicrocarrier. In some embodiments of any of these methods, the differentculturing method utilizes a different raw material, anti-clumping agent,or chemically-defined liquid culture medium. In some embodiments of anyof these methods, the method is used to perform high throughput cellculture experiments to perform a design-of-experiment (DOE) or aquality-by-design (QBD) study. In some embodiments of any of thesemethods, the shake tube is has a volume of between about 10 mL to about100 mL. In some embodiments of these methods, the mammalian cell issuspended in about 2 mL to about 20 mL of the first liquid culturemedium. In some embodiments of any of these methods, the mammalian cellis a Chinese hamster ovary (CHO) cell. In some embodiments of any ofthese methods, the recombinant protein is a secreted immunoglobulin, asecreted enzyme, a secreted growth factor, a secreted protein fragment,or a secreted engineered protein, and where the recombinant protein isrecovered from the first or second culture medium. In some embodimentsof any of these methods, the recombinant protein is recovered from themammalian cell. In some embodiments of any of these methods, therecombinant protein is an immunoglobulin, an enzyme, a growth factor, aprotein fragment, or an engineered protein. In some embodiments of anyof these methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium is performed simultaneously. In some embodiments of anyof these methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium is performed continuously. In some embodiments of any ofthese methods, the removing of the first volume of the first liquidculture medium and the adding of the second volume of the second liquidculture medium is performed periodically. In some embodiments of any ofthese methods, the first volume of the first liquid culture mediumremoved and the second volume of the second liquid culture medium addedare increased over time. In some embodiments of any of these methods,the first liquid culture medium and/or second liquid culture medium isselected from the group consisting of: a chemically-defined liquidculture medium, a serum-free liquid culture medium, a serum-containingliquid culture medium, an animal- derived component free liquid culturemedium, and a protein-free medium. In some embodiments of any of thesemethods, the shake tube incubated at a reactor angle of about 25 degreesto about 90 degrees from horizontal.

Also provided are methods of testing the efficacy of a first or secondliquid culture medium, a raw ingredient or supplement present in a firstor second liquid culture medium, or a source of a mammalian cell for usein a method of producing a recombinant protein. These methods include:providing a shake tube containing a mammalian cell disposed in a firstliquid culture medium, where the first liquid culture medium occupiesabout 10% to about 30% of the volume of the shake tube and contains aplurality of microcarriers at a concentration of about 1.0 g/L to about15.0 g/L; incubating the shake tube for a period of time at about 32° C.to about 39° C. and with a rotary agitation of about 120 revolutions perminute (RPM) to about 240 RPM; and after about the first 48 to 96 hoursof the period of time, continuously or periodically removing a firstvolume of the first liquid culture medium and adding to the first liquidculture medium a second volume of a second liquid culture medium, wherethe first and second volumes are about equal; detecting the recombinantprotein in the cell or in the first and/or second culture medium;comparing the amount of recombinant protein present in the cell or inthe first and/or second culture medium to a reference level ofrecombinant protein produced by a different method that uses one or moreof a different first or second liquid culture medium, a different rawingredient or supplement present in the first or second liquid culturemedium, or a different source of a mammalian cell; and identifying thefirst or second liquid culture medium, the raw ingredient or supplementpresent in the first or second liquid culture medium, or the source ofthe mammalian cell that is associated with an increased amount ofrecombinant protein as compared to the reference level as beingefficacious for use in a method of producing a recombinant protein. Insome embodiments of any of these methods, the shake tube is incubated ata reactor angle of about 25 degrees to about 90 degrees from horizontal.

Also provided are methods of optimizing a manufacturing process ofproducing a recombinant protein. These methods include: providing ashake tube containing a mammalian cell disposed in a first liquidculture medium, where the first liquid culture medium occupies about 10%to about 30% of the volume of the shake tube and contains a plurality ofmicrocarriers at a concentration of about 1.0 g/L to about 15.0 g/L;incubating the shake tube for a period of time at about 32° C. to about39° C. and with a rotary agitation of about 120 revolutions per minute(RPM) to about 240 RPM; and after about the first 48 to 96 hours of theperiod of time, continuously or periodically removing a first volume ofthe first liquid culture medium and adding to the first liquid culturemedium a second volume of a second liquid culture medium, where thefirst and second volumes are about equal; detecting the recombinantprotein in the cell or in the first and/or second culture medium;comparing the amount of recombinant protein present in the cell or inthe first and/or second culture medium to a reference level ofrecombinant protein produced by a different method; and identifying andremoving or altering in a manufacturing process any culture componentsor parameters that are associated with a decrease in the amount ofrecombinant protein produced as compared to the reference level, oridentifying and adding to a manufacturing process any culture componentsor parameters that are associated with an increase in the amount ofrecombinant protein produced as compared to the reference level. In someembodiments of any of these methods, the shake tube is incubated at areactor angle of about 25 degrees to about 90 degrees from horizontal.

Also provided are methods of testing for the presence of a contaminantin a first or second liquid culture medium, a raw material used togenerate a first or second liquid culture medium, or a source of amammalian cell. These methods include: providing a shake tube containinga mammalian cell disposed in a first liquid culture medium, where thefirst liquid culture medium occupies about 10% to about 30% of thevolume of the shake tube and contains a plurality of microcarriers at aconcentration of about 1.0 g/L to about 15.0 g/L; incubating the shaketube for a period of time at about 32° C. to about 39° C. and with arotary agitation of about 120 revolutions per minute (RPM) to about 240RPM; and after about the first 48 to 96 hours of the period of time,continuously or periodically removing a first volume of the first liquidculture medium and adding to the first liquid culture medium a secondvolume of a second liquid culture medium, where the first and secondvolumes are about equal; detecting the recombinant protein in the cellor in the first and/or second culture medium; comparing the amount ofrecombinant protein present in the cell or in the first and/or secondculture medium to a reference level of recombinant protein produced by adifferent method that uses one or more of a different first or secondliquid culture medium, a different raw material to generate the first orsecond liquid culture medium, or a different source of the mammaliancell; and identifying the first or second liquid culture medium, the rawmaterial used to generate the first or second liquid culture medium, orthe source of a mammalian cell as containing a contaminant when thelevel of recombinant protein produced is less than the reference level.In some embodiments of any of these methods, the contaminant is abiological contaminant. In some embodiments of any of these methods, thebiological contaminant is selected from the group of: mycobacterium, afungus, a bacterium, a virus, and an undesired mammalian cell. In someembodiments of any of these methods, the shake tube is incubated at areactor angle of about 25 degrees to about 90 degrees from horizontal.

As used herein, the word “a” or “plurality” before a noun represents oneor more of the particular noun. For example, the phrase “a mammaliancell” represents “one or more mammalian cells,” and the phrase“plurality of microcarriers” means “one or more microcarriers.”

The term “mammalian cell” means any cell from or derived from any mammal(e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow,or a rabbit). In some embodiments, the mammalian cell can be, e.g., animmortalized cell, a differentiated cell, or an undifferentiated cell.

The term “target cell density” means a specific concentration of cellsper volume of culture medium for producing a recombinant protein inculture. Target cell density can vary depending upon the specificmammalian cell cultured. For example, the target cell density can beabout 1.0×10⁶ cells/mL to about 50×10⁶ cells/mL (e.g., between about1.0×10⁶ cells/mL to about 3.0×10⁶ cells/mL, between about 1.0×10⁶cells/mL to about 2.0×10⁶ cells/mL, or between about 2.0×10⁶ cells/mL toabout 3.0×10⁶ cells/mL).

The term “substantially free” means a composition (e.g., a liquidculture medium) that is at least or about 90% free (e.g., at least orabout 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%free) of a specific substance (e.g., a mammalian cell or microcarriers).

The term “culturing” or “cell culturing” means the maintenance or growthof a mammalian cell in a liquid culture medium under a controlled set ofphysical conditions.

The term “shake tube” is meant a vessel (e.g., a sterile vessel) thatcan retain liquid culture medium that has at least one gas permeablesurface (e.g., an end that has at a gas-permeable element, e.g., amembrane, which may also act as a sterile barrier) and/or at least onevent cap, and is capable of retaining liquid culture medium within thevessel upon agitation (e.g., rotary agitation), and at least a portionof its shape is approximately cylindrical. For example, a shake tube canbe an Eppendorf™ tube (e.g., a 50-mL or 15-mL Eppendorf tube), or anyart-recognized equivalent or modified version thereof.

The term “liquid culture medium” means a fluid that contains sufficientnutrients to allow a mammalian cell to grow in the medium in vitro. Forexample, a liquid culture medium can contain one or more of: amino acids(e.g., 20 amino acids), a purine (e.g., hypoxanthine), a pyrimidine(e.g., thymidine), choline, inositol, thiamine, folic acid, biotin,calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin,pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron,copper, zinc, selenium, and other necessary trace metals, and sodiumbicarbonate. A liquid culture medium may contain serum from a mammal. Insome instances, a liquid culture medium does not contain serum oranother extract from a mammal (a defined liquid culture medium). Aliquid culture medium may contain trace metals, a mammalian growthhormone, and/or a mammalian growth factor. Non-limiting examples ofliquid culture medium are described herein and additional examples areknown in the art and are commercially available.

The phrase “substantially different type of liquid culture medium” meansa liquid culture medium that contains a substantially different nutrientprofile from another liquid culture medium. For example, a liquidculture medium that contains one or more of a mammalian serum, mammalianprotein, or a mammalian protein fraction or extract (e.g., aserum-containing liquid culture medium) is a substantially differenttype of liquid culture medium than one that does not contain any of amammalian serum, mammalian protein, or a mammalian protein fraction orextract (e.g., an animal-derived component free liquid culture medium, aserum-free liquid culture medium, a chemically-defined liquid culturemedium, and a protein-free liquid culture medium).

The phrase “substantially the same type of liquid culture medium” meansa liquid culture medium that contains about the same nutrient profile ascompared to another liquid culture medium. For example, if liquidculture medium A and liquid culture medium B both contain one or more ofa mammalian serum, mammalian protein, and a mammalian protein fractionor extract (e.g., a serum-containing liquid culture medium), there aresubstantially the same. In another example, if liquid culture medium Aand liquid culture medium B both do not contain any of a mammalianserum, mammalian protein, and a mammalian protein fraction or extract(e.g., an animal-derived component free liquid culture medium, aserum-free liquid culture medium, a chemically-defined liquid culturemedium, and a protein-free liquid culture medium), they aresubstantially the same.

The term “microcarrier” means a particle (e.g., an organic polymer) thathas a size of between 20 μm to about 1000 μm that contains a surfacethat is permissive or promotes attachment of a mammalian cell (e.g., anyof the mammalian cells described herein or known in the art). Amicrocarrier can contain one or more pores (e.g., pores with an averagediameter of about 10 μm to about 100 μm). Non-limiting examples ofmicrocarriers are described herein. Additional examples of microcarriersare known in the art. A microcarrier can contain, e.g., a polymer (e.g.,cellulose, polyethylene glycol, or poly-(lactic-co-glycolic acid)).

The term “animal-derived component free liquid culture medium” means aliquid culture medium that does not contain any components (e.g.,proteins or serum) derived from an animal.

The term “serum-free liquid culture medium” means a liquid culturemedium that does not contain animal serum.

The term “serum-containing liquid culture medium” means a liquid culturemedium that contains animal serum.

The term “chemically-defined liquid culture medium” means a liquidculture medium in which substantially all of the chemical components areknown. For example, a chemically-defined liquid culture medium does notcontain fetal bovine serum, bovine serum albumin, or human serumalbumin, as these preparations typically contain a complex mix ofalbumins and lipids.

The term “protein-free liquid culture medium” means a liquid culturemedium that does not contain any protein (e.g., any detectable protein).

“Rotary agitation” is a term well-known in the art and refers to themovement of a shake tube in a generally circular fashion, e.g.,clock-wise or counter-clockwise, in order to, e.g., increase thedissolved 02 concentration in a liquid culture medium contained therein.Agitation can be performed using any art-known method, e.g., aninstrument that moves the shake tube in a circular or ellipsoidalmotion, such as a rotary shaker. Exemplary devices that can be used toperform rotary agitation are described herein. Additional examples ofsuch devices are also known in the art and are commercially available.

The term “immunoglobulin” means a polypeptide containing an amino acidsequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60,70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., avariable domain sequence, a framework sequence, or a constant domainsequence). The immunoglobulin may, for example, include at least 15amino acids of a light chain immunoglobulin and/or at least 15 aminoacids of a heavy chain immunoglobulin. The immunoglobulin may be anisolated antibody (e.g., an IgG, IgE, IgD, IgA, or IgM). Theimmunoglobulin may be a subclass of IgG (e.g., IgG1, IgG2, IgG3, orIgG4). The immunoglobulin may be an antibody fragment, e.g., a Fabfragment, a F(ab')2 fragment, or an a scFv fragment. The immunoglobulinmay also be a bi-specific antibody or a tri-specific antibody, or adimer, trimer, or multimer antibody, or a diabody, an Affibody®, or aNanobody®. The immunoglobulin can also be an engineered proteincontaining at least one immunoglobulin domain (e.g., a fusion protein).Non-limiting examples of immunoglobulins are described herein andadditional examples of immunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion ofa polypeptide sequence that is at least or about 4 amino acids, e.g., atleast or about 5 amino acids, at least or about 6 amino acids, at leastor about 7 amino acids, at least or about 8 amino acids, at least orabout 9 amino acids, at least or about 10 amino acids, at least or about11 amino acids, at least or about 12 amino acids, at least or about 13amino acids, at least or about 14 amino acids, at least or about 15amino acids, at least or about 16 amino acids, at least or about 17amino acids, at least or about 18 amino acids, at least or about 19amino acids, or at least or about 20 amino acids in length, or more than20 amino acids in length. A recombinant protein fragment can be producedusing any of the methods described herein.

The term “engineered protein” means a polypeptide that is not naturallyencoded by an endogenous nucleic acid present within an organism (e.g.,a mammal). Examples of engineered proteins include enzymes (e.g., withone or more amino acid substitutions, deletions, insertions, oradditions that result in an increase in stability and/or catalyticactivity of the engineered enzyme), fusion proteins, antibodies (e.g.,divalent antibodies, trivalent antibodies, or a diabody), andantigen-binding proteins that contain at least one recombinantscaffolding sequence.

The term “recover” or “recovering” in certain contexts means at leastpartially purifying or isolating (e.g., at least or about 5%, e.g., atleast or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or at least or about 95% pure by weight) arecombinant protein from one or more other components present in thecell culture medium (e.g., mammalian cells or culture medium proteins)or one or more other components (e.g., DNA, RNA, or other proteins)present in a mammalian cell lysate. Non-limiting methods for recoveringa protein from a liquid culture medium or from a mammalian cell lysateare described herein and others are known in the art.

The term “secreted protein” or “secreted recombinant protein” means aprotein or a recombinant protein that originally contained at least onesecretion signal sequence when it is translated within a mammalian cell,and through, at least in part, enzymatic cleavage of the secretionsignal sequence in the mammalian cell, is released at least partiallyinto the extracellular space (e.g., a liquid culture medium).

The phrase “gradient perfusion” is art-known and refers to theincremental change (e.g., increase or decrease) in the volume of culturemedium removed and added to an initial culture volume over incrementalperiods (e.g., an about 24-hour period, a period of between about 1minute and about 24-hours, or a period of greater than 24 hours) duringthe culturing period (e.g., the culture medium re-feed rate on a dailybasis). The fraction of media removed and replaced each day can varydepending on the particular cells being cultured, the initial seedingdensity, and the cell density at a particular time.

The term “feed-batch culture” means the incremental or continuousaddition of a second liquid culture medium to an initial cell culturewithout substantial or significant removal of the first liquid culturemedium from the cell culture. In some instances, the second liquidculture medium is the same as the first liquid culture medium. In otherinstances, the second liquid culture medium is a concentrated form ofthe first liquid culture medium and/or is added as a dry powder.

The term “reactor angle” refers to the angle of deviation from thehorizontal position that the shake tube containing a mammalian cell isplaced during the culturing methods described herein. For example, whenthe shake tube containing a mammalian cell is a 50-mL conical tube andis standing vertical relative to the lab bench or ground, the reactorangle is 90°, and when the shake tube containing a mammalian cell is a50-mL conical tube and is placed horizontal relative to the lab bench orground, the reactor angle is 0°. In another example, when a shake tubecontaining a mammalian cell is a 50-mL conical tube and is placedequidistant between the vertical and horizontal positions (relative tothe lab bench or ground), the reactor angle is 45°.

“Specific productivity rate” or “SPR” as used herein refers to the massor enzymatic activity of a recombinant protein produced per mammaliancell per day. The SPR for a recombinant antibody is usually measured asmass/cell/day. The SPR for a recombinant enzyme is usually measured asunits/cell/day or (units/mass)/cell/day.

“Volume productivity rate” or “VPR” as used herein refers to the mass orenzymatic activity of recombinant protein produced per volume of culture(e.g., per L of bioreactor, vessel, or tube volume) per day. The VPR fora recombinant antibody is usually measured as mass/L/day. The VPR for arecombinant enzyme is usually measured as units/L/day or mass/L/day.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. Other features andadvantages of the invention will be apparent from the following detaileddescription and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph of the viable cell concentration over time in shaketube cell culture process runs performed using an agitation frequency of120 RPM (n=3) or 140 RPM (n=3), and in shake flask cell culture processruns (n=3). The mean of the data ±standard deviation are shown.

FIG. 2 is a graph of the viable cell concentration over time in shaketube cell culture process runs performed using an agitation frequency of140 RPM (n=3) and in shake flask cell culture process runs (n=3). Themean of the data ±standard deviation are shown.

FIG. 3 is a graph of the cumulative volumetric productivity (units/L)over time in shake tube cell culture process runs performed using anagitation frequency of 140 RPM (n=3) and in shake flask cell cultureprocess runs (n=3). The mean of the data ±standard deviation are shown.

FIG. 4 is a graph of the glucose consumption rate (grams ofglucose/L/day) over time in shake tube cell culture process runsperformed using an agitation frequency of 140 RPM (n=3) and in shakeflask cell culture process runs (n=3). The mean of the data ±standarddeviation are shown.

FIG. 5 is a graph of the lactate production rate (grams oflactate/L/day) over time in shake tube cell culture process runsperformed using an agitation frequency of 140 RPM (n=3) and in shakeflask cell culture process runs (n=3). The mean of the data ±standarddeviation are shown.

FIG. 6 is a graph of the glutamine consumption rate (mM/day) over timein shake tube cell culture process runs performed using an agitationfrequency of 140 RPM (n=3) and in shake flask cell culture process runs(n=3). The mean of the data ±standard deviation are shown.

FIG. 7 is a graph of the pH over time in shake tube cell culture processruns performed using an agitation frequency of 140 RPM (n=3) and inshake flask cell culture process runs (n=3). The mean of the data±standard deviation are shown.

FIG. 8 is a graph of the partial pressure of O₂ (pO₂) (mmHg) over timein shake tube cell culture process runs performed using an agitationfrequency of 140 RPM (n=3) and in shake flask cell culture process runs(n=3). The mean of the data ±standard deviation are shown.

FIG. 9 is a graph of the partial pressure of CO₂ (pCO₂) (mmHg) over timein shake tube cell culture process runs performed using an agitationfrequency of 140 RPM (n=3) and in shake flask cell culture process runs(n=3). The mean of the data ±standard deviation are shown.

DETAILED DESCRIPTION

Provided herein are methods of culturing a mammalian cell in a shaketube using a plurality of microcarriers and batch re-feed perfusion. Theculturing methods described herein can achieve high mammalian cellconcentration levels, thereby improving the overall efficiency of aculturing process and providing high yields of desirable cellularproducts, such as recombinant proteins. For example, the methods canprovide a viable mammalian cell concentration (e.g., in the first and/orsecond liquid culture medium, or the first and/or second liquid culturemedium in one or more of the first, second, and third time periods) ofgreater than 2×10⁶ cells per mL, greater than 3×10⁶ cells/mL, greaterthan 4×10⁶ cells/mL, greater than 5×10⁶ cells/mL, greater than 6×10⁶cells/mL, greater than 7×10⁶ cells/mL, greater than 8×10⁶ cells/mL,greater than 9×10⁶ cells/mL, greater than 10×10⁶ cells/mL, greater than12×10⁶ cells/mL, greater than 14×10⁶ cells/mL, greater than 16×10⁶cells/mL, greater than 18×10⁶ cells/mL, greater than 20×10⁶ cells/mL,greater than 25×10⁶ cells/mL, greater than 30×10⁶ cells/mL, greater than35×10⁶ cells/mL, greater than 40×10⁶ cells/mL, greater than 45×10⁶cells/mL, or greater than 50×10⁶ cells/mL. For example, the culturingmethod can result in a viable mammalian cell concentration of between1×10⁶ cells/mL and 3×10⁶ cells/mL, between 3×10⁶ cells/mL and 5×10⁶cells/mL, between 5×10⁶ cells/mL and 7×10⁶ cells/mL, between 7×10⁶cells/mL and 9×10⁶ cells/mL, between 9×10×10⁶ cells/mL and 11×10⁶cells/mL, between 10×10⁶ cells/mL and 12×10⁶ cells/mL, between 11×10⁶cells/mL and 13×10⁶ cells/mL, between 12×10⁶ cells/mL and 14×10⁶cells/mL, between 14×10⁶ cells/mL and 16×10⁶ cells/mL, between 16×10⁶cells/mL and 18×10⁶ cells/mL, between 18×10⁶ cells/mL and 20×10⁶cells/mL, between 20×10⁶ cells/mL and 25×10⁶ cells/mL, between 25×10⁶cells/mL and 30×10⁶ cells/mL, between 30×10⁶ cells/mL and 35×10⁶cells/mL, between 35×10⁶ cells/mL and 40×10⁶ cells/mL, between 40×10⁶cells/mL and 45×10⁶ cells/mL, between 45×10⁶ cells/mL and 50×10⁶cells/mL, or greater than 50×10⁶ cells/mL. In some instances, themethods described herein result in an increase in the biologicalactivity of a recombinant protein (e.g., as compared to the biologicalactivity of a recombinant protein produced by a different method).

A variety of different methods to determine the cell density or viablecell density can be used, and are well-known in the art. For example, asample of the cell culture containing microcarriers can be treated torelease the cells from the surface of the microcarriers, and thereleased cells can optionally be diluted in physiological buffer, andthe cell suspension (e.g., diluted cell suspension) placed in ahemocytometer and counted using light microscopy. In another method, theviable cell density can be determined using a similar method, butincluding in the physiological buffer a dye that is selectively taken upby non-viable cells (e.g., trypan blue, such as Vi-CELL method fromBeckman Coulter (see Beckman Coulter website)). In yet another example,the cell density or viable cell density can be determined usingfluorescence-assisted flow cytometry (e.g., GUAVA from Merck Millipore(see Millipore website), and other cell counting methods.

In some instances, the culturing method results in a significantlyimproved specific productivity rate. For example, the specificproductivity rate achieved by the methods provided herein can be atleast 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold,90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold,160-fold, 170-fold, 180-fold, 190-fold, or 200-fold greater than thespecific productivity rate achieved using an art-known culturing method(e.g., a different shake tube culture method). The volume productivityrate achieved by the present methods can be at least 200 units/L/day, atleast 300 units/L/day, at least 400 units/L/day, at least 500units/L/day, at least 600 units/L/day, at least about 800 units/L/day,at least about 1,000 units/L/day, at least about 1,200 units/L/day, atleast about 1,400 units/L/day, at least about 1,600 units/L/day, atleast about 1,800 units/L/day, at least about 2,000 units/L/day, atleast about 2,200 units/L/day, at least about 2,400 units/L/day, atleast about 2,600 units/L/day, at least about 2,800 units/L/day, atleast about 3,000 units/L/day, at least 4,000 units/L/day, at least5,000 units/L/day, at least 6,000 units/L/day, at least 7,000units/L/day, at least 8,000 units/L/day, at least 9,000 units/L/day, atleast 10,000 units/L/day, higher than 1,000 units/L/day, or higher than10,000 units/L/day (e.g., in the first and/or second liquid culturemedium, or the first and/or second liquid culture medium used in one ormore of the first, second, and third time period). In some embodiments,the productivity achieved by the present methods can be at least 0.1g/L, at least 0.2 g/L, at least 0.5 g/L, at least 0.75 g/L, at least1.0g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, at least 3.5 g/L, at least4.0 g/L, at least 4.5 g/L, or at least 5.0 g/L (e.g., in the firstand/or second liquid culture medium, or the first and/or second liquidculture medium used in the first, second, and third time period).

The biological activity of a recombinant protein can be assessed using avariety of methods known in the art, and will depend on the activity ofthe specific recombinant protein. For example, the biological activityof a recombinant protein that is an immunoglobulin (e.g., an antibody oran antibody fragment) can be determined by measuring the affinity of theantibody to bind to its specific epitope (e.g., using Biocore orcompetitive enzyme-linked immunosorbent assays). The recombinant proteinmay be an enzyme (e.g., a recombinant galactosidase, e.g., a recombinanthuman alpha-galactosidase) and the biological activity may be determinedby measuring the enzyme's activity (e.g., determining the catalytic rateconstant of the enzyme by measuring a decrease in the concentration of adetectable substrate or an increase in the concentration of a detectableproduct (e.g., using spectrophotometry or light emission). For example,the biological activity of a recombinant galactosidase can be detectedby measuring a decrease in the level of globotriasylceramide (GL-3) orgalabiosylceramide, or an increase in the level of ceramide dihexosideor galactose.

Methods of Culturing a Mammalian Cell

In a method that is exemplary of those described herein, a shake tube isprovided. A first liquid culture medium is added to the shake tube suchthat the medium occupies, e.g., about 10% to about 30% (e.g., about 10%to about 20%, about 20% to about 30%, about 10% to about 12%, about 12%to about 14%, about 14% to about 16%, about 16% to about 18%, about 18%to about 20%, about 20% to about 22%, about 22% to about 24%, about 24%to about 26%, about 26% to about 28%, about 28% to about 30%, about 10%to about 15%, about 15% to about 20%, about 20% to about 25%, or about25% to about 30%) of the volume of the shake tube. At least onemammalian cell and a plurality of microcarriers (a final concentrationin the shake tube of about 1.0 g/L to about 15.0 g/L, e.g., a finalconcentration in the shake tube of between about 1.0 g/L to about 2.5g/L, about 1.0 g/L to about 2.0 g/L, about 1.0 g/L to about 1.75 g/L,about 1.0 g/L to about 1.5 g/L, about 1.0 g/L to about 1.25 g/L, about2.5 g/L to 5.0 g/L, about 5.0 g/L to about 7.5 g/L, about 7. 5 g/L toabout 10.0 g/L, about 10.0 g/L to about 12.5 g/L, about 12.5 g/L toabout 15.0 g/L, about 1.0 g/L to about 5.0 g/L, about 5.0 g/L to about10.0 g/L, about 10.0 g/L to about 15.0 g/L, about 2.5 g/L to about 3.5g/L, about 3.0 g/L to about 4.0 g/L, about 4.0 g/L to about 5.0 g/L,about 5.0 g/L to about 6.0 g/L, about 6.0 g/L to about 7.0 g/L, about7.0 g/L to about 8.0 g/L, about 8.0 g/L to about 9.0 g/L, about 9.0 g/Lto about 10.0 g/L, about 10.0 g/L to about 11.0 g/L, about 11.0 g/L toabout 12.0 g/L, about 12.0 g/L to about 13.0 g/L, about 13.0 g/L toabout 14.0 g/L, or about 14.0 g/L to about 15.0 g/L) is added to thefirst liquid culture medium, i.e., either before the medium is added tothe shake tube or afterward. As one skilled in the art can appreciate,the steps of the addition of the liquid culture medium, a mammaliancell, and the liquid culture medium to the shake tube can occur in anyorder. The shake tube is incubated for a period of time at about 32° C.to about 39° C. (e.g., 32° C. to 34° C., 32° C. to 37° C., 34° C. to 37°C., 37° C. to 39° C.) and agitated, e.g., on a rotary shaking device, atabout 120 RPM to about 240 RPM (e.g., about 120 RPM to about 230 RPM,about 120 RPM to about 220 RPM, about 120 RPM to about 210 RPM, about120 RPM to about 200 RPM, about 120 RPM to about 190 RPM, about 120 RPMto about 180 RPM, about 120 RPM to about 170 RPM, about 120 RPM to about160 RPM, about 120 RPM to about 150 RPM, about 130 RPM to about 180 RPM,about 130 RPM to about 170 RPM, about 140 RPM to about 170 RPM, about150 RPM to about 170 RPM, about 120 RPM to about 140 RPM, about 130 RPMto about 150 RPM, about 140 RPM to about 160 RPM, about 150 RPM to about170 RPM, about 160 RPM to about 180 RPM, about 160 RPM to about 220 RPM,about 160 RPM to about 210 RPM, about 150 RPM to about 190 RPM, or about180 RPM to about 210 RPM). The cells can be incubated, for example, inan incubator, such as a shake incubator with throw (orbit) diameter of25 mm or from about 3 mm to about 50 mm, while changing the RPMaccordingly. After the first 48 to 96 hours of the period of time ofincubation, continuously or periodically over the period of time, afirst volume of the first liquid culture medium (e.g., containing anymammalian cell concentration, e.g., a first volume of first liquidculture medium which is or is made substantially free of mammalian cellsand/or microcarriers) is removed, and a second volume of a second liquidculture medium is added to the first liquid culture medium. Typically,the first and the second volumes are roughly equal, but can vary by asmall amount, e.g., by up to about 10% when the first and second volumesare compared. In some embodiments, the second volume of the secondliquid culture medium added is less (e.g., at most about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or 10% less) or more (e.g., at most about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% more) than the first volume of thefirst liquid culture medium removed. As is known in the art, the termincubating can include short periods of time (e.g., between 10 secondsand about10 minutes, between 10 seconds and about 20 minutes, between 10seconds and about 30 minutes, between 10 seconds and about 40 minutes,between about 10 seconds and about 50 minutes, or between 10 seconds andabout 1 hour) in which a shake tube containing the mammalian cell andliquid culture medium is removed from an incubator in order to removethe first volume of the first liquid culture medium and add the secondvolume of the second liquid culture medium. In some embodiments, anautomatic sampler can be employed to remove the first volume of thefirst culture medium and add the second volume of the second liquidculture medium to the shake tube while the shake tube remains in theincubator.

In another exemplary method, a shake tube is first provided. A firstliquid culture medium is added to the shake tube such that the mediumoccupies, e.g., about 10% to about 30% (e.g., about 10% to about 20%,about 20% to about 30%, about 10% to about 12%, about 12% to about 14%,about 14% to about 16%, about 16% to about 18%, about 18% to about 20%,about 20% to about 22%, about 22% to about 24%, about 24% to about 26%,about 26% to about 28%, about 28% to about 30%, about 10% to about 15%,about 15% to about 20%, about 20% to about 25%, or about 25% to about30%) of the volume of the shake tube. At least one mammalian cell and aplurality of microcarriers (a final concentration in the shake tube ofabout 1.0 g/L to about 15.0 g/L, e.g., a final concentration in theshake tube of between about 1.0 g/L to about 2.5 g/L, about 1.0 g/L toabout 2.0 g/L, about 1.0 g/L to about 1.75 g/L, about 1.0 g/L to about1.5 g/L, about 1.0 g/L to about 1.25 g/L, about 2.5 g/L to 5.0 g/L,about 5.0 g/L to about 7.5 g/L, about 7.5 g/L to about 10.0 g/L, about10.0 g/L to about 12.5 g/L, about 12.5 g/L to about 15.0 g/L, about 1.0g/L to about 5.0 g/L, about 5.0 g/L to about 10.0 g/L, about 10.0 g/L toabout 15.0 g/L, about 2.5 g/L to about 3.5 g/L, about 3.0 g/L to about4.0 g/L, about 4.0 g/L to about 5.0 g/L, about 5.0 g/L to about 6.0 g/L,about 6.0 g/L to about 7.0 g/L, about 7.0 g/L to about 8.0 g/L, about8.0 g/L to about 9.0 g/L, about 9.0 g/L to about 10.0 g/L, about 10.0g/L to about 11.0 g/L, about 11.0 g/L to about 12.0 g/L, about 12.0 g/Lto about 13.0 g/L, about 13.0 g/L to about 14.0 g/L, or about 14.0 g/Lto about 15.0 g/L) is added to the first liquid culture medium, i.e.,either before the medium is added to the shake tube or afterward. Asnoted above, the addition of the liquid culture medium, a mammaliancell, and the liquid culture medium to the shake tube can occur in anyorder. Then, in a first time period, the shake tube is incubated atabout 35° C. to about 39° C. (e.g., 35° C. to 37° C., 36° C. to 39° C.,or 37° C. to 39° C.) with a rotary agitation of about 120 RPM to about240 RPM (e.g., about 120 RPM to about 230 RPM, about 120 RPM to about220 RPM, about 120 RPM to about 210 RPM, about 120 RPM to about 200 RPM,about 120 RPM to about 190 RPM, about 120 RPM to about 180 RPM, about120 RPM to about 170 RPM, about 120 RPM to about 160 RPM, about 120 RPMto about 150 RPM, about 130 RPM to about 180 RPM, about 130 RPM to about170 RPM, about 140

RPM to about 170 RPM, about 150 RPM to about 170 RPM, about 120 RPM toabout 140 RPM, about 130 RPM to about 150 RPM, about 140 RPM to about160 RPM, about 150 RPM to about 170 RPM, about 160 RPM to about 180 RPM,about 160 RPM to about 220 RPM, about 160 RPM to about 210 RPM, about150 RPM to about 190 RPM, or about 180 RPM to about 210 RPM). The cellscan be incubated, for example, in an incubator, such as a shakeincubator with throw (orbit) diameter from about 3 mm to about 50 mm.After about the first 48 to 96 hours of the first time period, in eachsubsequent 24-hour period, (i) continuously or periodically removing afirst volume of the first liquid culture medium that is substantiallyfree of microcarriers from the shake tube, wherein the first volume isabout 10% to about 95% (e.g., about 10% to 20%, about 20% to about 30%,about 30% to about 40%, about 40% to about 50%, about 50% to about 60%,about 60% to about 70%, about 70% to about 80%, about 80% to about 95%,about 50% to about 95%, about 50% to about 90%, or about 60% to about90%) of the volume of the first liquid culture medium; and (ii) addingto the shake tube a second volume of a second liquid culture medium,wherein the first and second volume are about equal. As noted above, thefirst and the second volumes are roughly equal, but can vary by a smallamount, e.g., by up to about 10% when the first and second volumes arecompared. Once the cell concentration reaches about target cell density(e.g., about 1.0×10⁶ cells/mL, about 1.5×10⁶ cells/mL, about 2.0×10⁶cells/mL, about 2.2×10⁶ cells/mL, about 2.4×10⁶ cells/mL, about 2.6×10⁶cells/mL, about 2.8×10⁶ cells/mL, about 3.0×10⁶ cells/mL, about 3.2×10⁶cells/mL, about 3.4×10⁶ cells/mL, about 3.6×10⁶ cells/mL, about 3.8×10⁶cells/mL, about 4.0×10⁶ cells/mL, about 1.0×10⁶ cells/mL to 4.0×10⁶cells/mL, about 2.0×10⁶ cells/mL to about 4.0×10⁶ cells/mL, about2.0×10⁶ cells/mL to about 4.0×10⁶ cells/mL, about 4.0×10⁶ cells/mL toabout 6.0×10⁶ cells/mL, about 6.0×10⁶ cells/mL to about 8.0×10⁶cells/mL, about 8.0×10⁶ cells/mL to about 10.0×10⁶ cells/mL, about10.0×10⁶ cells/mL to about 15.0×10⁶ cells/mL, about 15.0×10⁶ to about20.0×10⁶ cells/mL, about 20.0×10⁶ cells/mL to about 25.0×10⁶ cells/mL,about 25.0×10⁶ cells/mL to about 30.0×10⁶ cells/mL, about 30.0×10⁶cells/mL to about 35.0×10⁶ cells/mL, about 35.0×10⁶ cells/mL to about40.0×10⁶ cells/mL, about 40.0×10⁶ cells/mL to about 45.0×10⁶ cells/mL,or about 45.0×10⁶ cells/mL to about 50.0×10⁶ cells/mL) the shake tube isincubated for a second time period of about 2 days to about 7 days(e.g., about 2 days to about 4 days, about 3 days to about 5 days, about4 days to about 6 days, and about 5 days to about 7 days), at about 32°C. to about 39° C. (e.g., about 32° C. to about 35° C., about 32° C. toabout 37° C., about 32° C. to about 38° C., about 34° C. to about 39°C., about 34° C. to about 37° C., about 35° C. to about 38° C., about35° C. to about 39° C., about 36° C. to about 39° C., or about 37° C. toabout 39° C.) with the rotary agitation, and in each 24-hour period,performing steps (i) and (ii) described above, where the first andsecond liquid culture media used in the first time period are of asubstantially different type from those used in the second time period.Then, in a third period of time of greater than 2 days (e.g., 2, 3, 4,5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100 days, greater than 100 days, 110 days, 120 days, 130days, 140 days, 150 days, 160 days, 170 days, 180 days, 190 days, or 200days, or at most 100 days, 125 days, 150 days, 175 days, 200 days, 225days, 250 days, 275 days, or 300 days) incubating the shake tube atabout 35° C. to about 39° C. (e.g., about 35° C. to about 37°, 36° C. toabout 38° C., about 37° C. to about 39° C., or about 36° C. to about 39°C.) with the rotary agitation, and in each 24-hour period, performingsteps (i) and (ii) listed above, where the first and second liquidculture media used in the second time period are of the same type asthose used in the third time period.

Various non-limiting examples of each aspect of these culturing methodsare described below. The exemplary aspects of the methods providedherein can be used in any combination without limitation.

Mammalian Cells

The methods provided herein can be used to culture a variety ofdifferent mammalian cells. In some examples of all the methods describedherein, the mammalian is an adherent cell. Non-limiting examples ofmammalian cells that can be cultured using any of the methods describedherein include: Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells,CHO-K1s cells, Sp2.0, myeloma cells (e.g., NS/0), B-cells, hybridomacells, T-cells, human embryonic kidney (HEK) cells (e.g, HEK 293E andHEK 293F), African green monkey kidney epithelial cells (Vero) cells,and Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK)cells. Additional mammalian cells that can be cultured using the methodsdescribed herein are known in the art.

The mammalian cell can contain a recombinant nucleic acid (e.g., anucleic acid stably integrated in the mammalian cell's genome) thatencodes a recombinant protein (e.g., a recombinant protein that issecreted by the mammalian cell). Non-limiting examples of recombinantnucleic acids that encode exemplary recombinant proteins are describedbelow, as are recombinant proteins that are producible using the methodsdescribed herein. In some instances, the mammalian cell disposed in theshake tube for culturing is derived from a larger culture. For example,the mammalian cell in the shake tube can be derived from a large-scalebioreactor culture, i.e., a satellite culture can be prepared using anyof the methods described herein.

Culture Media

Liquid culture media are known in the art. The first and/or secondtissue culture medium (e.g., the first and second liquid culture mediumused in the first time period or the second and third time periods) canbe supplemented with a mammalian serum (e.g., fetal calf serum andbovine serum), and/or a growth hormone or growth factor (e.g., insulin,transferrin, and epidermal growth factor). Alternatively or in addition,the first and/or second liquid culture medium (e.g., the first and/orsecond liquid culture medium, e.g., the first and/or second liquidculture medium in the first time period or the second and third timeperiods) can be a chemically-defined liquid culture medium, ananimal-derived component free liquid culture medium, a serum-free liquidculture medium, or a serum-containing liquid culture medium.Non-limiting examples of chemically-defined liquid culture media,animal-derived component free liquid culture media, serum-free liquidculture media, and serum-containing liquid culture media arecommercially available.

A liquid culture medium typically contains an energy source (e.g., acarbohydrate, such as glucose), essential amino acids (e.g., the basicset of twenty amino acids plus cysteine), vitamins and/or other organiccompounds required at low concentrations, free fatty acids, and/or traceelements. The first and/or second liquid culture medium (e.g., the firstand/or second liquid culture medium used in the first time period or thesecond and third time periods) can, if desired, be supplemented with,e.g., a mammalian hormone or growth factor (e.g., insulin, transferrin,or epidermal growth factor), salts and buffers (e.g., calcium,magnesium, and phosphate salts), nucleosides and bases (e.g., adenosine,thymidine, and hypoxanthine), protein and tissue hydrolysates, and/orany combination of these or other additives.

Non-limiting examples of liquid culture media that are particularlyuseful in the presently described methods include, e.g., CD CHO, OptiCHO, and Forti CHO (all available from Life Technologies; Grand Island,NY), Hycell CHO medium (Thermo Fisher Scientific, Inc.; Waltham, MA),Ex-cell CD CHO Fusion medium (Sigma-Aldrich Co.; St. Louis, Mo.), andPowerCHO medium (Lonza Group, Ltd.; Basel, Switzerland). Mediumcomponents that also may be useful in the present methods include, butare not limited to, chemically-defined (CD) hydrolysates, e.g., CDpeptone, CD polypeptides (two or more amino acids), and CD growthfactors. Additional examples of liquid tissue culture medium and mediumcomponents are known in the art.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium described herein can be thesame type of media or different type of media. For example, in examplesof the methods that include a first time period, a second time period,and a third time period, the first and second liquid culture medium usedin the first time period are substantially different from the first andsecond liquid culture medium used in the second and third time period,and the first and second liquid culture medium used in the second andthird time period are substantially the same. For example, the first andsecond liquid culture medium used in the first time period can beselected from the group consisting of a serum-containing liquid culturemedium or a liquid culture medium that contains a mammalian protein or amammalian protein fraction or extract, and the first and second liquidculture medium used in the second and third time periods can be selectedfrom the group of: an animal-derived component free liquid culturemedium, a serum-free liquid culture medium, a chemically-defined liquidculture medium, and a protein-free liquid culture medium.

Microcarriers

In the methods described herein, a plurality of microcarriers is addedto the liquid culture medium (e.g., the first and/or second liquidculture medium). For example, the plurality of microcarriers can have anaverage diameter of between about 20 μm to about 1 mm (e.g., betweenabout 20 μm and about 250 μm, between about 100 μm to about 250 μm,between about 150 μm to about 250 μm, between about 250 μm and 500 μm,between about 200 μm to about 300 μm, between about 750 μm and 1 mm,between about 200 μm to about 800 μm, between about 200 μm and about 500μm, or between about 500 μm and about 800 μm), where the microcarriershave a surface that is permissive or promotes attachment of a mammaliancell (e.g., any of the mammalian cells described herein or known in theart). In some examples, a microcarrier can contain one or more pores(e.g., one or more pores with an average diameter of about 10 μm toabout 100 μm (e.g., between about 10 μm and 20 μm, about 20 μm to about30 μm, about 30 μm to about 40 μm, about 50 μm to about 60 μm, about 60μm to about 70 μm, about 70 μm to about 80 μm, about 80 μm to about 90μm, about 90 μm to about 100 μm, about 10 μtm to about 45 μm, about 45μm to about 80 μm, about 25 μM to about 35 μm, or about 30 μm)). In someembodiments, the surface of the plurality of microcarriers and/or thesurface of the one or more pores in the plurality of microcarriers arecoated with an agent that promotes the attachment of a mammalian cell tothe microcarrier (e.g., attachment to the outer surface of themicrocarriers and/or the surface of the pores in the microcarrier).Examples of such agents that can be used to promote the attachment of amammalian cell include, but are not limited to, gelatin, collagen,poly-L-ornithine, polystyrene, and laminin.

In some examples, the microcarriers have an average effective cellbinding surface area of between about 0.5 m²/g dry and 2.0 m²/g dry(e.g., between about 0.75 m²/g dry and 1.25 m²/dry, between about 1.0m²/g dry and about 1.5 m²/dry, between about 1.25 m²/dry and about 1.5m²/dry, between about 1.5 m²/dry and about 2.0 m²/dry, and about 1.1m²/dry). In some examples, the microcarriers have an average volume ofabout 10 mL/g dry to about 70 mL/g dry (e.g., about 10 mL/g dry to about20 mL/g dry, about 20 mL/g dry to about 30 mL/g dry, about 30 mL/g dryto about 40 mL/g dry, about 40 mL/g dry to about 50 mL/g dry, about 50mL/g dry to about 60 mL/g dry, about 60 mL/g dry to about 70 mL/g dry,about 10 mL/g dry to about 40 mL/g dry, about 30 mL/g dry to about 40mL/g dry, about 40 mL/g dry to about 70 mL/g dry, or about 40 mL/g dry).In some embodiments, the average relative density of the microcarriersis between 0.8 g/mL to about 1.2 g/mL (e.g., about 0.8 g/mL to about 0.9g/mL, about 0.9 g/mL to about 1.0 g/mL, about 1.0 g/mL to about 1.1g/mL, about 1.0 g/mL, about 1.1 g/mL to about 1.2 g/mL, about 0.95 g/mLto about 1.05 g/mL, or about 1.03 g/mL).

In some embodiments, the microcarriers are approximately spherical orellipsoidal in shape. In other examples, the microcarriers have anabraded or rough surface with small protuberances that increase thetotal outer surface area of the microcarrier. In some embodiments, themicrocarriers have a network structure. In some examples, themicrocarriers are hygroscopic. In some examples, the microcarrierscontain cellulose.

In some embodiments, the microcarriers have an outer surface and/or themicrocarrier pores have a surface that is positively charged (e.g.,positively charged due to the presence of N,N-diethylaminoethyl groups).In some examples, the microcarriers have a network or net-like orweb-like structure. The microcarriers can have an average charge densityof about 0.5 meq/g to about 2.5 meq/g (e.g., about 0.5 meq/g to about1.5 meq/g, about 0.75 meq/g to about 1.25 meq/g, about 1.1 meq/g, about1.5 meq/g to about 2.5 meq/g, about 1.5 meq/g to about 2.0 meq/g, about1.8 meq/g, about 0.5 meq/g to about 1.0 meq/g, or about 1.0 meq/g toabout 1.5 meq/g).

In some instances, the microcarrier can contain a natural polymer and/ora synthetic polymer. Non-limiting examples of synthetic polymers includepolyethylene glycol (PEG), polyethylene oxide, polyethyleneimine,diethyleneglycol, triethyleneglycol, polyalkalene glycol, polyalkalineoxide, polyvinyl alcohol, sodium polyphosphate, polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,polymethacrylamide, polydimethylacrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyglycerine,polyaspartamide, polyoxyethlene-polyoxypropylene copolymer (poloxamer),carboxylic acids (e.g., acrylic acid, methacrylic acid, itaconic acid,and maleic acid), polyoxyethylenes, polyethyleneoxide, unsaturatedethylenic monodicarboxylic acids, polylactic acid (PLA), polypropyleneoxide, poly(lactide-co-glycolide) (PLGA), poly(epsilon-caprolactone),poly(ethylethylene), polybutadiene, polyglycolide, polymethylacrylate,polyvinylbutylether, polystyrene, polycyclopentadienylmethylnorbornene,polyethylenepropylene, polyethylethylene, polyisobutylene, polysiloxane,methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate,isobutyl acrylate, 2-ethyl acrylate, t-butyl acrylate, methacrylates(e.g., ethyl methacrylate, n-butyl methacrylate, and isobutylmethacrylate), acrylonitriles, methacrylonitrile, vinyls (e.g., vinylacetate, vinylversatate, vinylpropionate, vinylformamide,vinylacetamide, vinylpyridines, and vinylimidazole), aminoalkyls (e.g.,aminoalkylacrylates, aminoalkylsmethacrylates, andaminoalkyl(meth)acrylamides), styrenes, polyalkalene glycol,polyalkaline oxide, and lactic acids. Non-limiting examples of naturalpolymers include cellulose, lecithin, and hyaluronic acid. Amicrocarrier can contain a mixture of different polymers (e.g., anycombination of one or more polymers described herein or known in theart) in the same or different ratios. Any of the microcarriers describedherein can contain a core containing one or more polymers (e.g., any ofthe polymers described herein or known in the art) and an outer layerthat contains one or more different polymers (e.g., any of the polymersdescribed herein or known in the art). A plurality of microcarriers caninclude a combination of two or more different types of microcarriers(e.g., two or more microcarriers having a different shape, size, charge,or composition).

Non-limiting exemplary microcarriers that can be used in any of themethods described herein include CytoPore™ 1 and CytoPore™ 2 (availablefrom GE Healthcare, Life Sciences, Piscataway, N.J.). Additionalexamples of microcarriers that can be used in any of the methodsdescribed herein are publicly available and known in the art.

Shake Tubes

The shake tube can be sterile and have a volume between about 2 mL toabout 500 mL (e.g., a 2-mL, 4-mL, 5-mL, 10-mL, 15-mL, 20-mL, 25-mL,50-mL, 75-mL, 100-mL, 150-mL, 200-mL, 250-mL, 300-mL, 350-mL, 400-mL,450-mL, or 500-mL shake tube. The shake tube can have a volume, forexample, of about 2 mL to about 200 mL, about 10 mL to about 200 mL,about 2 mL to about 100 mL, about 20 mL to about 200 mL, about 20 mL toabout 100 mL, about 2 mL to about 15 mL, about 2 mL to about 25 mL,about 2 mL to about 50 mL, about 10 mL to about 50 mL, about 5 mL toabout 25 mL, about 25 mL to about 50 mL, about 2 mL to about 15 mL,about 3 mL to about 20 mL, about 3 mL to about 15 mL, about 2 mL toabout 250 mL, about 2 mL to about 300 mL, about 2 mL to about 400 mL,about 2 mL to about 450 mL, about 10 mL to about 250mL, about 10 mL toabout 350 mL, about 10 mL to about 400 mL, about 10 mL to about 450 mL,about 20 mL to about 250 mL, about 20 mL to about 350 mL, about 20 mL toabout 400 mL, about 20 mL to about 450 mL, about 50 mL to about 100 mL,about 50 mL to about 150 mL, about 50 mL to about 250 mL, about 50 mL toabout 300 mL, about 50 mL to about 350 mL, about 50 mL to about 400 mL,about 50 mL to about 450 mL, about 100 mL to about 150 mL, about 150 mLto about 200 mL, about 200 mL to about 250 mL, about 250 mL to about 350mL, about 300 mL to about 400 mL, or about 350 mL to about 450 mL.Non-limiting examples of shake tubes are Tubespin® shake tubes (TPPTechno Plastic Products AG, Trasadingen, Switzerland).

The shake tube can include at least one gas permeable surface (e.g., atleast one surface having a gas permeable membrane which may also act asa sterile barrier) and/or at least one vented cap. A shake tube may haveon its outer surface a structure that allows the shake tube to be stablyplaced in a tissue culture incubator (e.g., a rotary incubator).

The interior surface of the shake tube may have at least one coating(e.g., at least one coating of gelatin, collagen, poly-L-ornithine,polystyrene, and laminin). The shake tube can be, for example, aTubeSpin® shake tube available from Techno Plastic Products AG,Trasadingen, Switzerland, the shake tubes available from Sartorius, AG,Germany, and sterile Becton Dickinson (BD) Falcon tubes. Additionalexamples of shake tubes (e.g., different shapes and dimensions of shaketubes) and interior surface coatings of shake tubes are known in the artand can be used in the present methods.

Agitation

The methods described herein involve the agitation of the culturecontaining the mammalian cell, a plurality of microcarriers, and thefirst and/or second liquid culture medium. The agitation can occur at afrequency of at about 120 RPM to about 240 RPM, (e.g., about 125 RPM toabout 180 RPM, about 125 RPM to about 175 RPM, about 130 RPM to about180 RPM, about 130 RPM to about 170 RPM, about 135 RPM to about 170 RPM,about 135 RPM to about 165 RPM, about 140 RPM to about 165 RPM, about140 RPM to about 160 RPM, about 130 RPM to about 170 RPM, about 120 RPMto about 150 RPM, about 125 RPM to about 155 RPM, about 130 RPM to about160 RPM, about 140 RPM to about 170 RPM, about 145 RPM to about 175 RPM,about 150 RPM to about 180 RPM, about 120 RPM to about 150 RPM, about150 RPM to about 180 RPM, about 120 RPM to about 230 RPM, about 120 RPMto about 220 RPM, about 120 RPM to about 210 RPM, about 120 RPM to about200 RPM, about 120 RPM to about 190 RPM, about 130 RPM to about 200 RPM,or about 120 RPM to about 180 RPM) (e.g., in an incubator, such as ashake incubator with throw (orbit) diameter from about 3 mm to about 50mm).

As can be appreciated in the art, the level of agitation (e.g., RPMspeed) can be varied depending upon the size and shape of the shake tube(e.g., the diameter of the shake tube), the throw (orbit) diameter ofthe incubator that is used to perform the agitation, and the averagesize, shape, density, and concentration of the plurality ofmicrocarriers. For example, a smaller throw (orbit) diameter can requirea higher level of agitation (e.g., a higher RPM speed), while a largerthrow (orbit) diameter can require a lower level of agitation (e.g., alower RPM speed) to achieve the conditions necessary to achieve optimalviable cell density and recombinant protein production. For example, fora throw (orbit) diameter of, e.g., 9 mm, the frequency of agitation canbe greater than 180 RPM (e.g., between about 180 RPM to about 240 RPM).In another example, a shake tube having a larger diameter can require alower RPM speed, while a shake tube having a smaller diameter canrequire a higher RPM speed to achieve the conditions necessary toachieve optimal viable cell density and recombinant protein production.The frequency of agitation can be varied depending on cell cultureconditions, e.g., the concentration, density, and/or the size and/orsurface shape of the microcarriers. As one skilled in the art canappreciate, if microcarriers present in the first and/or second liquidculture medium (e.g., the first and/or second liquid culture medium usedin the first, second, and third time periods) have a high mass, a highdensity, a large outer surface area, or a relatively high velocity, thesheer forces generated by such microcarriers can have a negative impacton cell viability and recombinant protein production in the culture. Inaddition, those in the art can appreciate that the rate of agitationshould be high enough to avoid substantial and/or undesirable settlingof the microcarriers on the bottom of shake tube.

In some embodiments, the incubating is performed using a rotaryincubator with a throw (orbit) diameter of between about 25 mm to about50 mm and an agitation of between about 120 RPM to about 180 RPM (e.g.,about 125 RPM to about 180 RPM, about 125 RPM to about 175 RPM, about130 RPM to about 180 RPM, about 130 RPM to about 170 RPM, about 135 RPMto about 170 RPM, about 135 RPM to about 165 RPM, about 140 RPM to about165 RPM, about 140 RPM to about 160 RPM, about 130 RPM to about 170 RPM,about 120 RPM to about 150 RPM, about 125 RPM to about 155 RPM, about130 RPM to about 160 RPM, about 140 RPM to about 170 RPM, about 145 RPMto about 175 RPM, about 150 RPM to about 180 RPM, about 120 RPM to about150 RPM, or about 150 RPM to about 180 RPM). In some embodiments, theincubating is performed using a rotary incubator with a throw (orbit)diameter of about 3 mm to about 25 mm and an agitation of about 120 RPMto about 240 RPM (e.g., about 125 RPM to about 180 RPM, about 125 RPM toabout 175 RPM, about 130 RPM to about 180 RPM, about 130 RPM to about170 RPM, about 135 RPM to about 170 RPM, about 135 RPM to about 165 RPM,about 140 RPM to about 165 RPM, about 140 RPM to about 160 RPM, about130 RPM to about 170 RPM, about 120 RPM to about 150 RPM, about 125 RPMto about 155 RPM, about 130 RPM to about 160 RPM, about 140 RPM to about170 RPM, about 145 RPM to about 175 RPM, about 150 RPM to about 180 RPM,about 120 RPM to about 150 RPM, about 150 RPM to about 180 RPM, about120 RPM to about 230 RPM, about 120 RPM to about 220 RPM, about 120 RPMto about 210 RPM, about 120 RPM to about 200 RPM, about 120 RPM to about190 RPM, about 130 RPM to about 200 RPM, or about 120 RPM to about 180RPM).

Agitation can be performed, e.g., using rotary circular shaking at afrequency of about 120 RPM to about 240 RPM (e.g., about 125 RPM toabout 180 RPM, about 125 RPM to about 175 RPM, about 130 RPM to about180 RPM, about 130 RPM to about 170 RPM, about 135 RPM to about 170 RPM,about 135 RPM to about 165 RPM, about 140 RPM to about 165 RPM, about140 RPM to about 160 RPM, about 130 RPM to about 170 RPM, about 120 RPMto about 150 RPM, about 125 RPM to about 155 RPM, about 130 RPM to about160 RPM, about 140 RPM to about 170 RPM, about 145 RPM to about 175 RPM,about 150 RPM to about 180 RPM, about 120 RPM to about 150 RPM, about150 RPM to about 180 RPM, about 120 RPM to about 230 RPM, about 120 RPMto about 220 RPM, about 120 RPM to about 210 RPM, about 120 RPM to about200 RPM, about 120 RPM to about 190 RPM, about 130 RPM to about 200 RPM,or about 120 RPM to about 180 RPM). Alternatively or in addition, theshake tube can be agitated using a rotary ellipsoidal shaking, orhorizontal and/or vertical tilting of the shake tube. The agitation canbe performed continuously or periodically.

The agitation can be performed using a humidified atmosphere controlledincubator (e.g., at a humidity of about or greater than 20%, e.g., aboutor greater than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, ora humidity of 100%) with a mechanical device that provides the agitationof one or more of the shake tubes containing the mammalian cell, theplurality of microcarriers, and a liquid culture medium (e.g., the firstand/or second liquid culture medium, and the first and/or second liquidculture medium used in one or more of the first, second, and third timeperiods).

Reactor Angle

The shake tube can be incubated at a reactor angle of about 25 degreesto about 90 degrees (e.g., about 25 degrees to about 55 degrees, about25 degrees to about 90 degrees, about 35 degrees to about 90 degrees,about 45 degrees to about 90 degrees, or about 35 to about 65 degrees)from horizontal. For example, the shake tube can be placed at a reactorangle of about 60 degrees to about 85 degrees from horizontal, about 70degrees to about 85 degrees from horizontal, about 25 degrees to about60 degrees, about 25 degrees to about 55 degrees, about 30 degrees toabout 55 degrees from horizontal, about 40 degrees to about 55 degreeshorizontal, or about 40 degrees to about 50 degrees from horizontal. Theshake tube may be placed at a reactor angle of about 45 degrees fromhorizontal to about 50 degrees from horizontal, or from about 40 degreesfrom horizontal to about 45 degrees from horizontal. The shake tube maybe placed in a device that specifically and securely positions the shaketube at a reactor angle of about 25 degrees to about 90 degrees fromhorizontal (e.g., specifically positions the container at a reactorangle of about 25 degrees to about 90 degrees, about 35 degrees to about90 degrees, about 45 degrees to about 90 degrees, about 35 degrees toabout 65 degrees, or about 40 degrees to about 55 degrees fromhorizontal). The positioning of the shake tube can be performed usingany means known in the art, e.g., through the use of a brace or alocking element.

Temperature

The culturing methods described herein can be performed at a temperatureof about 32° C. to about 39° C. For example, in some methods the shaketube can be incubated at a temperature of about 37° C. from thebeginning to the end of the culture run. Some examples of the methodsdescribed herein include a first time period during which the shake tubeis incubated at a temperature of about 35° C. to about 39° C., a secondtime period during which the shake tube is incubated at about 32° C. toabout 39° C., and third time period during which the shake tube isincubated at about 35° C. to about 39° C. Skilled practitioners willappreciate that the temperature can be changed at specific time point(s)in the culturing method (e.g., during one or more of the first timeperiod, the second time period, and the third time period), e.g., on anhourly or daily basis. For example, the temperature can be changed orshifted (e.g., increased or decreased) at about one day, two days, threedays, four days, five days, six days, seven days, eight days, nine days,ten days, eleven days, twelve days, fourteen days, fifteen days, sixteendays, seventeen days, eighteen days, nineteen days, or about twenty daysor more after the initial seeding of the shake tube with the mammaliancell) or at any time point within the first, second, and/or third timeperiods described herein. For example, the temperature can be shiftedupwards (e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0° C.). For example, thetemperature can be shifted downwards (e.g., a change of up to or about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10°C.).

Culture Medium Removal and Replacement

The methods described herein include removing from the shake tube afirst volume of a first liquid culture medium (e.g., containing anyconcentration of mammalian cells and any recombinant protein, e.g., afirst volume of a first liquid culture medium that is substantially freeof cells and/or microcarriers), and adding to the shake tube a secondvolume of a second liquid culture medium, wherein the first volume andthe second volume are about equal. Removal and adding can be performedsimultaneously or sequentially, or a combination of the two. Further,removal and adding can be performed continuously (e.g., at a rate thatremoves and replaces a volume of between 0.1% to 800% (e.g., between 1%and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%,between 1% and 350%, between 1% and 300%, between 1% and 250%, between1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and50%, between 15% and 40%, between 8% and 80%, and between 4% and 30%) ofthe volume of the shake tube or the first liquid culture medium volumeover any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, five times a day, or more thanfive times a day), or any combination thereof. Where performedperiodically, the volume that is removed or replaced (e.g., within abouta 24-hour period, within an incremental time period of about 0.1 hour toabout 24 hours, or within an incremental time period of greater than 24hours) can be, e.g., between 0.1% to 800% (e.g., between 1% and 700%,between 1% and 600%, between 1% and 500%, between 1% and 400%, between1% and 300%, between 1% and 200%, between 1% and 100%, between 100% and200%, between 5% and 150%, between 10% and 50%, between 15% and 40%,between 8% and 80%, and between 4% and 30%) of the volume of the shaketube or the first liquid culture medium volume. The first volume of thefirst liquid culture medium removed and the second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 0.1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the firstvolume of the first liquid culture medium is removed (volume/unit oftime) and the rate at which the second volume of the second liquidculture medium is added (volume/unit of time) can be varied. The rate atwhich the first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can be aboutthe same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 0.1 hour and about 24 hours or an incrementaltime period of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the shake tube volume or the firstliquid culture medium volume to about 25% to about 150% of the shaketube volume or the first liquid culture medium volume.

In some examples of the methods described herein, after the first 48 to96 hours of the culture period, in each 24-hour period, the first volumeof the first liquid culture medium removed (e.g., in the first, second,and/or third time period) and the second volume of the second liquidculture medium added (e.g., in the first, second, and/or third timeperiod) is about 10% to about 95% (e.g., about 10% to about 20%, about10% to about 20%, about 20% to about 30%, about 30% to about 40%, about40% to about 50%, about 50% to about 60%, about 60% to about 70%, about70% to about 80%, about 80% to about 90%, about 30% to about 80%, about85% to about 95%, about 60% to about 80%, or about 70%) of the volume ofthe first liquid culture medium.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be substantially different. In someembodiments that include a first time period, second time period, andthird time period, the first and second liquid culture media used in thefirst time period are a substantially different type of media comparedto the first and second liquid culture media used in the second timeperiod, and the first and second liquid culture media used in the secondtime period are the same type of media compared to the first and secondliquid culture media used in the third time period. As can be recognizedin the art, the first and second liquid culture media used in the firsttime period do not have to be exactly the same (as long as they are thesame type of culture medium); any of the first and second liquid culturemedia used in the second time period and/or third time period do nothave to be exactly the same (again, as long as they are the same type ofmedium and a substantially different media type from the first andsecond liquid culture medium used in the first time period).

The first volume of the first liquid culture medium can be removed,e.g., by centrifuging (e.g., slow-speed swinging bucket centrifugation)the shake tube or using any other automated system, and removing thefirst volume of the first liquid culture (e.g., a first volume of thefirst liquid culture medium that is substantially free of cells and/ormicrocarriers) from the supernatant. Alternatively or in addition, thefirst volume of the first liquid culture medium can be removed byseeping or gravity flow of the first volume of the first liquid culturemedium through a sterile membrane with a molecular weight cut-off thatexcludes the mammalian cell and/or microcarriers. Alternatively or inaddition, the first volume of the first liquid culture medium can beremoved by stopping or significantly decreasing the rate of agitationfor a period of at least 10 seconds (e.g., at least 30 seconds, 40seconds, 50 seconds, 1 minutes, 2 minutes, 3 minutes, 4 minutes, 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40minutes, 50 minutes, or 1 hour) and removing or aspirating the firstvolume of the first liquid culture medium from the top of the shake tube(e.g., removal from a part of the liquid culture medium where themicrocarriers have not settled due to gravitational force). The shaketube may be placed in an incubator during the period in which theagitation is ceased. One skilled in the art will understand that theshake tube may be removed from the incubator for a short period of time(e.g., less than 30 minutes, 20 minutes, 15 minutes, 10 minutes, 8minutes, 6 minutes, 4 minutes, 2 minutes, or 1 minute) while the firstliquid culture medium is removed from the shake tube.

The second volume of the second liquid culture medium can be added tothe first liquid culture medium, e.g., by perfusion pump. The secondliquid culture medium can be added to the first liquid culture mediummanually (e.g., by pipetting the second volume of the second liquidculture medium directly onto the first liquid culture medium) or in anautomated fashion.

In some instances, removing the first volume of the first liquid culturemedium (e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells and/or microcarriers) and addingto the first liquid culture medium a second volume of the second liquidculture medium does not occur within at least 1 hour (e.g., within 2hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours,within 7 hours, within 8 hours, within 9 hours, within 10 hours, within12 hours, within 14 hours, within 16 hours, within 18 hours, within 24hours, within 36 hours, within 48 hours, within 72 hours, within 96hours, or after 96 hours) of the seeding of the shake tube with amammalian cell.

CO₂

Methods described herein can further include incubating the shake tubein an atmosphere containing at most or about 1% to 15% CO₂ (e.g., atmost or about 14% CO₂, 12% CO₂, 10% CO₂, 8% CO₂, 6% CO₂, 5% CO₂, 4% CO₂,3% CO₂, 2% CO₂, or at most or about 1% CO₂). Moreover, any of themethods described herein can include incubating the shake tube in ahumidified atmosphere (e.g., at least or about 20%, 30%, 40%, 50%, 60%,70%, 85%, 80%, 85%, 90%, or at least or about 95% humidity, or about100% humidity).

Exemplary Devices

Non-limiting examples of devices that can be used to perform theculturing methods described herein include: Appropriate TechnicalResources (Maryland, USA) distributes INFORS Multiron shake incubator(INFORS; Basel, Switzerland), and Kuhner shake incubator (Kuhner AG;Basel, Switzerland). Non-limiting examples of devices that can be usedto perform the culturing methods include a rotary incubator with a throw(orbit) diameter of between about 3 mm to about 50 mm (e.g., betweenabout 1 mm and about 25 mm, or between about 25 mm and about 50 mm).Additional examples of shake incubators are known in the art.

Methods of Producing a Recombinant Protein

Also provided herein are methods of producing a recombinant protein,which include culturing a cell that is capable of producing therecombinant protein using a method described herein. Followingperformance of the method, the recombinant protein can be recovered fromthe mammalian cell (e.g., the mammalian cell that is attached to themicrocarrier) and/or from the first or second culture medium (e.g., thefirst and/or second liquid culture medium used in one or more of thefirst, second, and third time periods). In some embodiments, therecombinant protein is recovered from the first and/or second liquidculture medium at any given time point during the culturing method(e.g., recovered from the first and/or second liquid culture medium onone or more of days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 of culture,or after more than 100 days of culture, or at any time point during oneor more of the first time period, the second time period, and the thirdtime period).

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., shake tubes, volumes, rates or frequencies ofreplacing culture volumes, agitation frequencies, type of microcarrier,temperatures, media, and CO₂ concentrations) can be used in anycombination to perform these methods. Further, any of the mammaliancells described herein or known in the art can be used to produce arecombinant protein.

A nucleic acid encoding a recombinant protein can be introduced into amammalian cell using a wide variety of methods known in molecularbiology and molecular genetics. Non-limiting examples includetransfection (e.g., lipofection), transduction (e.g., lentivirus,adenovirus, or retrovirus infection), and electroporation. In someinstances, the nucleic acid that encodes a recombinant protein is notstably integrated into a chromosome of the mammalian cell (transienttransfection), while in others the nucleic acid is integrated.Alternatively or in addition, the nucleic acid encoding a recombinantprotein can be present in a plasmid and/or in a mammalian artificialchromosome (e.g., a human artificial chromosome). Alternatively or inaddition, the nucleic acid can be introduced into the cell using a viralvector (e.g., a lentivirus, retrovirus, or adenovirus vector). Thenucleic acid can be operably linked to a promoter sequence (e.g., astrong promoter, such as a β-actin promoter and CMV promoter, or aninducible promoter). A vector containing the nucleic acid can, ifdesired, also contain a selectable marker (e.g., a gene that confershygromycin, puromycin, or neomycin resistance to the mammalian cell).

In some instances, the recombinant protein is a secreted protein and isreleased by the mammalian cell into the extracellular medium (e.g., thefirst and/or second liquid culture medium, e.g., the first and/or secondliquid culture medium used in one or more of the first, second, andthird time periods). For example, a nucleic acid sequence encoding asoluble recombinant protein can contain a sequence that encodes asecretion signal peptide at the N- or C-terminus of the recombinantprotein, which is cleaved by an enzyme present in the mammalian cell,and subsequently released into the extracellular medium (e.g., the firstand/or second liquid culture medium, or the first and/or second liquidculture medium used in one or more of the first, second, and third timeperiods). In other instances, the recombinant protein is a solubleprotein that is not secreted, and the recombinant protein is recoveredfrom within the mammalian cell (e.g., from within the mammalian cellthat is attached to the microcarrier, e.g., recovered from the mammaliancell attached to the microcarrier after it has been unattached from themicrocarrier).

Non-limiting examples of recombinant proteins that can be produced bythe methods provided herein include immunoglobulins (including light andheavy chain immunoglobulins, antibodies, or antibody fragments (e.g.,any of the antibody fragments described herein), enzymes (e.g., agalactosidase (e.g., an alpha-galactosidase),

Myozyme, or Cerezyme), proteins (e.g., human erythropoietin, tumornecrosis factor (TNF), or an interferon alpha or beta), or immunogenicor antigenic proteins or protein fragments (e.g., proteins for use in avaccine). In some embodiments, the recombinant protein is an engineeredantigen-binding polypeptide that contains at least one multifunctionalrecombinant protein scaffold (see, e.g., the recombinant antigen-bindingproteins described in Gebauer et al., Current Opin. Chem. Biol.13:245-255, 2009; and U.S. Patent Application Publication No.2012/0164066 (herein incorporated by reference in its entirety)).Non-limiting examples of recombinant proteins that are antibodiesinclude: panitumumab, omalizumab, abagovomab, abciximab, actoxumab,adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab,alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab,apolizumab, atinumab, tocilizumab, basilizimab, bectumomab, belimumab,bevacizumab, biciromab, canakinumab, cetuximab, daclizumab, densumab,eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab,etaracizumab, golimumab, infliximab, natalizumab, palivizumab,panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, andtrastuzumab. Additional examples of therapeutic antibodies that can beproduced by the methods described herein are known in the art.Additional non-limiting examples of recombinant proteins that can beproduced by the present methods include: alglucosidase alfa, laronidase,abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidasebeta, interferon beta-la, darbepoetin alfa, tenecteplase, etanercept,coagulation factor IX, follicle stimulating hormone, interferon beta-la,imiglucerase, dornase alfa, epoetin alfa, and alteplase.

A secreted, soluble recombinant protein can be recovered from the liquidculture medium (e.g., the first and/or second liquid culture medium,e.g., the first and/or second liquid culture medium used in one or moreof the first, second, and third time periods) by removing or otherwisephysically separating the liquid culture medium from microcarriers andtheir associated mammalian cells. A variety of different methods forremoving liquid culture medium from mammalian cells are known in theart, including, for example, centrifugation, filtration, pipetting,and/or aspiration. The secreted recombinant protein can then berecovered and further purified from the liquid culture medium using avariety of biochemical techniques including various types ofchromatography (e.g., affinity chromatography, molecular sievechromatography, cation exchange chromatography, or anion exchangechromatography) and/or filtration (e.g., molecular weight cut-offfiltration).

To recover an intracellular recombinant protein, the mammalian cell(e.g., the mammalian cell attached to the microcarrier) can be lysed. Insome examples, the mammalian cell is released from the surface of themicrocarrier before it is lysed. Methods for releasing an adherent cellfrom the surface of a microcarrier are known in the art (e.g., vortexingor agitation). In other examples, the mammalian cell is lysed while itis still attached to the microcarrier (e.g., using any of the exemplarymethods listed below).

A wide variety of methods for lysing mammalian cells are known in theart, including, for example, sonication and/or detergent, enzymatic,and/or chemical lysis. A recombinant protein can be purified from amammalian cell lysate using a variety of biochemical methods known inthe art, typically starting with a step of centrifugation to remove thecellular debris, and then one or more additional steps (e.g., one ormore types of chromatography (e.g., affinity chromatography, molecularsieve chromatography, cation exchange chromatography, or anion exchangechromatography) and/or filtration (e.g., molecular weight cut-offfiltration)).

In some embodiments, the recovered recombinant protein is at least orabout 50% pure by weight, e.g., at least or about 55% pure by weight, atleast 60% pure by weight, at least 65% pure by weight, at least 70% pureby weight, at least 75% pure by weight, at least 80% pure by weight, atleast 85% pure by weight, at least 90% pure by weight, at least 95% pureby weight, at least 96% pure by weight, at least 97% pure by weight, atleast 98% pure by weight, or at least or about 99% pure by weight, orgreater than 99% pure by weight.

In some embodiments, the recovered recombinant protein is a recombinanthuman protein that has one or more different biophysical properties ascompared to the same native protein in a human (e.g., differences in thetype or amount of glycosylation, differences in phosphorylation,differences in acylation, differences in metallation or metalstoichiometry, and/or differences in cofactor binding).

Also provided herein is a recombinant protein produced by any of themethods described herein.

Methods for Testing a Manufacturing Process

Also provided herein are methods for testing a manufacturing process formaking a recombinant protein. These methods include performing a methodof producing a recombinant protein described herein and, during themethod and/or afterward, detecting or measuring at least one (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, or twelve)culture readout (e.g., the recombinant protein in the cell or in thefirst and/or second culture medium (e.g., the first and/or second liquidculture medium used in one or more of the first, second, and third timeperiods), glucose consumption, viable cell concentration, lactateproduction, volumetric productivity, specific productivity, lactateyield from glucose, glutamine concentration, glutamate concentration, pHof culture medium, partial pressure or concentration of dissolved CO₂,partial pressure or concentration of dissolved 02, metabolite masstransfer, and metabolite mass balance); and comparing the at least oneculture readout to a reference level of the at least one (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, or twelve)culture readout (e.g., a reference level of the recombinant protein inthe cell or in the first and/or second culture medium (e.g., the firstand/or second liquid culture medium used in one or more of the first,second, and third time periods), glucose consumption, viable cellconcentration, lactate production, volumetric productivity, specificproductivity, lactate yield from glucose, glutamine concentration,glutamate concentration, pH of culture medium, concentration or partialpressure of dissolved CO₂, concentration or partial pressure ofdissolved O₂, metabolite mass transfer, and metabolite mass balance).

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., shake tubes, volumes, type of microcarrier, rates orfrequencies of replacing culture volumes, agitation frequencies,temperatures, media, and CO₂ exposure) described herein can be used inany combination to perform these methods. Further, any of the mammaliancells described herein or known in the art can be used in the methods.

The reference level of the at least one culture readout (e.g., level ofrecombinant protein in the cell or in the first and/or second culturemedium (e.g., the first and/or second liquid culture medium used in oneor more of the first, second, and third time periods), glucoseconsumption, viable cell concentration, lactate production, volumetricproductivity, specific productivity, lactate yield from glucose,glutamine concentration, glutamate concentration, pH of culture medium,concentration or partial pressure of dissolved CO₂, concentration orpartial pressure of dissolved O₂, metabolite mass transfer, andmetabolite mass balance) can be a level produced using a differentculturing method, e.g., a culturing method that utilizes at least onedifferent culture parameter (e.g., a different first and/or secondliquid culture medium (e.g., a different first and/or second liquidculture medium in one or more of the first, second, or third timeperiods), a different mammalian cell, a different frequency and/or typeof agitation, a different type or concentration of microcarrier, adifferent batch re-feed or perfusion rate (e.g., 10% to 95% of the shaketube volume or the first liquid culture medium volume over each 24-hourtime period after the first 48 to 96 hours of culture), and any of theother culture parameters described herein).

The methods described herein can be used to test the effect of anycomponent or feature of a manufacturing process. For example, the methoddescribed herein can be used to test the effect of different rawmaterials, microcarriers, agitation levels, shake tubes, anti-clumpingagents, culture media (e.g., chemically-defined culture media), ornutrient elements or compounds on the at least one culture readout(e.g., any of the culture readouts described herein, e.g., the effect onrecombinant protein production and/or mammalian cell growth). Forexample, provided herein are methods of testing the efficacy of a firstor second liquid culture medium, a raw ingredient or supplement presentin a first or second liquid culture medium, or a source of a mammaliancell for use in a method of producing a recombinant protein that includeproviding a shake tube containing a mammalian cell disposed in a firstliquid culture medium, wherein the first liquid culture medium occupies,e.g., about 10% to about 30% of the volume of the shake tube, andcontains a plurality of microcarriers at a concentration of about 1.0g/L to about 15.0 g/L; incubating the shake tube for a period of time atabout 32° C. to about 39° C. and with a rotary agitation of about 120revolutions per minute (RPM) to about 240 RPM; and after about the first48 to 96 hours of the period of time, continuously or periodicallyremoving a first volume of the first liquid culture medium and adding tothe first liquid culture medium a second volume of a second liquidculture medium, wherein the first and second volumes are about equal;detecting or determining at least one culture readout (e.g., any of theculture readouts described herein, e.g., the recombinant protein in thecell or in the first and/or second culture medium); comparing the atleast one culture readout to a reference level of the at least oneculture readout (e.g., any of the culture readouts described herein,e.g., recombinant protein in the cell or in the first and/or secondliquid culture medium) produced by a different culturing method thatuses one or more of a different first or second liquid culture medium, adifferent raw ingredient or supplement present in the first or secondliquid culture medium, or a different source of a mammalian cell; andidentifying the first or second liquid culture medium, the rawingredient or supplement present in the first or second liquid culturemedium, or the source of the mammalian cell that is associated withbeneficial change (e.g., increase or decrease) in the at least oneculture readout (e.g., an increased amount of recombinant protein) ascompared to the reference level as being efficacious for use in a methodof producing a recombinant protein. For example, an increase inrecombinant protein level, an increase in viable cell concentration, anincrease in volumetric productivity, and an increase in glucoseconsumption compared to the reference level indicates that the first orsecond liquid culture medium, the raw ingredient or supplement presentin a first or second liquid culture medium, or the source of themammalian cell are efficacious for use in a method of producing arecombinant protein.

The methods described herein can also be used to test the effect ofchanging any of the various cell culture parameters described herein orknown in the art (e.g., the volume or shape of a shake tube, thefrequency of agitation, the sheer force generated by the plurality ofmicrocarriers in the first and/or second liquid culture medium, theculture seeding density, the pH of the first and/or second liquidculture medium (e.g., the pH of the first and/or second liquid culturemedium used in one or more of the first, second, or third time periods),dissolved 02 concentration or partial pressure, the inner surfacecoating of the shake tube, one or more of the concentration, size,shape, surface properties, density, and porosity of the microcarriers,the various ingredients within a liquid culture medium (e.g., the firstand/or second liquid culture medium, e.g., the first and/or secondliquid culture medium used in one or more of the first, second, andthird time periods), the amount and/or type of agitation, the mammaliancell type or line, dissolved CO₂ concentration or partial pressure, thetemperature, the volume of liquid culture medium (e.g., the volume ofthe first and/or second liquid culture medium), and/or the rate orfrequency of removing the first volume of the first liquid culturemedium and adding the second volume of the second liquid culture mediumto the first culture medium (e.g., the rate or frequency of removing thefirst volume of the first culture medium and adding the second volume ofthe second liquid culture medium in one or more of the first, second,and third time periods). The methods can also be used to test thequality of water used to prepare the liquid culture medium (e.g., thefirst and/or second liquid culture medium, e.g., the first and/or secondliquid culture medium used in one or more of the first, second, andthird time periods) and/or the effect of different trace metals in theliquid culture medium on at least one culture readout on at least oneculture readout (e.g., any of the culture readouts described herein,e.g., the effect on recombinant protein production and/or mammalian cellgrowth). The methods can also be used to test the effect of a growthfactor or growth hormone (e.g., the effect of the presence of a growthfactor or growth hormone in the first time period) on at least oneculture readout (e.g., any of the culture readouts described herein,e.g., the effect on recombinant protein production and/or mammalian cellgrowth). The method can also be used to test filtration processes andfilters used to prepare the first and/or second liquid culture medium(e.g., the first and/or second liquid culture medium used in one or moreof the first, second, and third time periods). The method can also beused to test liquid culture medium stability and the effect of a liquidculture medium on at least one culture readout (e.g., any of the culturereadouts described herein, e.g., the effect on recombinant proteinproduction and/or mammalian cell growth). The method can also be used toscreen various recombinant cells lines and cell banks for their abilityto produce a desired recombinant protein (e.g., a desired secretedtherapeutic protein). As noted herein, the method can also be used toscreen any cell culture process parameter, including but limited to, thetype and frequency of agitation, sheer force generated by themicrocarriers, perfusion rate and volume, culture seeding density, andothers.

The method described herein can also be used to test for the presence ofa contaminant in a first or second liquid culture medium, a raw materialused to generate a first or second liquid culture medium, or a source ofa mammalian cell. For example, provided herein are methods of testingfor the presence of a contaminant in a first or second liquid culturemedium, raw materials used to generate a first or second liquid culturemedium, or a source of a mammalian cell that include providing a shaketube containing a mammalian cell disposed in a first liquid culturemedium, wherein the first liquid culture medium occupies about, e.g.,10% to about 30% of the volume of the shake tube, and contains aplurality of microcarriers at a concentration of about 1.0 g/L to about15.0 g/L; incubating the shake tube for a period of time at about 32° C.to about 39° C. and with a rotary agitation of about 120 revolutions perminute (RPM) to about 240 RPM; and after about the first 48 to 96 hoursof the period of time, continuously or periodically removing a firstvolume of the first liquid culture medium and adding to the first liquidculture medium a second volume of a second liquid culture medium,wherein the first and second volumes are about equal; detecting ordetermining at least one culture readout (e.g., any of the culturereadouts described herein, e.g., the recombinant protein in the cell orin the first and/or second culture medium); comparing the at least oneculture readout to a reference level of the at least one culture readout(e.g., any of the culture readouts described herein, e.g., amount ofrecombinant protein present in the cell or in the first and/or secondculture medium) produced by a different culturing method that uses oneor more of a different first or second liquid culture medium, differentraw materials to generate the first or second liquid culture medium, ora different source of the mammalian cell; and identifying the first orsecond liquid culture medium, the raw materials used to generate thefirst or second liquid culture medium, or the source of a mammalian cellas containing a contaminant when the level of the at least one cultureparameter is detrimentally changed (e.g., increased or decreased)compared to the reference level. For example, a decrease in recombinantprotein production (e.g., a decrease in recombinant protein in the cellor in the first and/or second culture medium), volumetric productivity,or viable cell concentration as compared to the reference level is adetrimental change that indicates the presence of a contaminant in thefirst or second liquid culture medium, a raw material used to generatethe first or second liquid culture medium, or the source of themammalian cell. Some methods further include one or more assays todetermine the identity of the contaminant present in the first or secondliquid culture medium, the raw material used to generate the first orsecond liquid culture medium, or the source of the mammalian cell. Thecontaminant can be a biological contaminant (e.g., a mycobacterium, afungus, a bacterium, a virus, or an undesired mammalian cell). Forexample, the contaminant can be a vesivirus. The contaminant can be aninorganic contaminant. The contaminant can also be a physicallyuncharacterized substance.

The methods can used to conduct high throughput cell culture experimentsto perform a design-of-experiment (DOE) or a quality-by-design (QBD)optimization of cell culturing methods. For example, provided herein aremethods of optimizing a manufacturing process of producing a recombinantprotein that include providing a shake tube containing a mammalian celldisposed in a first liquid culture medium, wherein the first liquidculture medium occupies, e.g., about 10% to about 30% of the volume ofthe shake tube, and contains a plurality of microcarriers at aconcentration of about 1.0 g/L to about 15.0 g/L; incubating the shaketube for a period of time at about 32° C. to about 39° C. and with arotary agitation of about 120 revolutions per minute (RPM) to about 240RPM; and after about the first 48 to 96 hours of the period of time,continuously or periodically removing a first volume of the first liquidculture medium and adding to the first liquid culture medium a secondvolume of a second liquid culture medium, wherein the first and secondvolumes are about equal; detecting at least one culture readout (e.g.,any of the culture readouts described herein, e.g., amount ofrecombinant protein in the cell or in the first and/or second culturemedium); comparing the at least one culture readout to a reference levelof the at least one culture readout (e.g., any of the culture readoutsdescribed herein, e.g., amount of recombinant protein present in thecell or in the first and/or second culture medium) produced by adifferent culturing method; and identifying and removing or altering ina manufacturing process any culture components or parameters that areassociated with a detrimental change (e.g., increase or decrease) in theat least one culture readout (e.g., any of the culture readoutsdescribed herein, e.g., amount of recombinant protein produced) ascompared to the reference level of the at least one culture readout(e.g, any of the culture readouts described herein, e.g., recombinantprotein produced), or identifying and adding to a manufacturing processany culture components or parameters that are associated with abeneficial change (e.g., increase or decrease) in the at least oneculture readout (e.g., any of the culture readouts described herein,e.g., amount of recombinant protein produced) as compared to thereference level of the at least one culture readout (e.g., any of theculture readouts described herein, e.g., recombinant protein produced).For example, an increase in the amount of recombinant protein produced,volumetric productivity, specific productivity, or viable cellconcentration is a beneficial change in a culture readout, and adecrease in the amount of recombinant protein produced, volumetricproductivity, specific productivity, or viable cell concentration is adetrimental change in a culture readout. In some instances, the methodis used to identify in a high throughput fashion, optimized cell cultureconditions that can be used for up-scaled (e.g., bioreactor) productionof a recombinant protein.

In any of the methods described in this section, the reference level ofthe at least one culture readout can be from a larger-scale culture(e.g., a perfusion bioreactor, e.g., a 2000-L perfusion bioreactor, 40-Lperfusion bioreactor, or a 12-L perfusion bioreactor). In someembodiments of any of the methods described in this section, themammalian cell is cultured in a shake tube using any of the methodsdescribed herein over the same time period that a larger-scale cultureis performed (cultured in parallel). For example, the inoculum used toinoculate the shake tube in any of the methods described herein is alsoused to inoculate a larger-scale perfusion bioreactor at approximatelythe same time.

In one embodiment, the inoculum that is used to seed the shake tube isobtained from a larger-scale culture (e.g., a larger-scale perfusionbioreactor). For example, an aliquot from a larger-scale culture (e.g.,an aliquot from a larger-scale perfusion bioreactor) is removed from thelarger-scale culture at any time point (e.g., removed during the growthphase, the transition phase, or the harvest phase described herein) andused to inoculate the shake tube (e.g., used to start a satellite shaketube culture). An aliquot can be removed from the larger-scale cultureduring the growth phrase and used to inoculate or seed a shake tubecontaining a liquid culture medium and a plurality of microcarriers(e.g., as described herein), and the shake tube is then incubated underconditions that replicate or are similar to the growth phase conditionsemployed in the larger-scale culture. An aliquot can alternatively, oradditionally, be removed from the larger-scale culture during thetransition phase and used to inoculate or seed a shake tube containing aliquid culture medium and a plurality of microcarriers (e.g., asdescribed herein), and the shake tube is then incubated under conditionsthat replicate or are similar to the transition phase conditionsemployed in the larger-scale culture. An aliquot can alternatively, oradditionally, be removed from the larger-scale culture during theharvest phase and used to inoculate or seed a shake tube containing aliquid culture medium and a plurality of microcarriers (e.g., asdescribed herein), and the shake tube is then incubated under conditionsthat replicate or are similar to the harvest phase conditions employedin the larger-scale culture. In any of these methods, one or moreculture parameters can be altered in the methods used to culture themammalian cell in the shake tube (as compared to the culture parametersor components used to culture the mammalian cell in the larger- scaleculture), at least one culture readout is measured (e.g., one or more ofany of the culture readouts described herein), and the at least oneculture readout is compared to the at least one culture readoutdetermined for the larger-scale culture. As can be appreciated by thosein the art, these methods can be used to test the effect of a specificculture parameter or component on at least one culture readout duringone or more specific phases in the culturing process (e.g., the effectof one or more culture parameters and/or culture component(s) on atleast one culture readout during the growth, transition, and/or harvestphase).

In certain embodiment, these methods can also be performed to determinewhether a contaminant is present in the larger-scale bioreactor, bydetermining or detecting at least one culture readout in the shake tubeculture (e.g., one or more of any of the culture readouts describedherein), comparing the at least one culture readout to a reference levelof the at least one culture readout (e.g., a level of the at least oneculture readout from a culture that is substantially free ofcontamination), and identifying the larger-scale bioreactor ascontaining a contaminant when the at least one culture readout in theshake tube culture as compared to the reference level of the at leastone culture readout indicates that a contaminant is present in the shaketube. The contaminant can be, for example, a biological contaminant,such as a virus, a fungus, an undesired mammalian cell, or a bacterium,such as a mycobacterium. The contaminant can be, for example, avesivirus.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Exemplary Culture Methods using a Shake Tube and Microcarriers

Human α-galactosidase can be produced using established recombinantengineering techniques in a CHO cell line. The current manufacturingproduction of recombinant human α-galactosidase utilizes a 2000-Lcontinuous perfusion microcarrier cell culture process technology.Typically the production cell culture process includes three phases: agrowth, transition, and harvest phase. There is a demand for a highthroughput cell culture process system that would accurately model thecell culture process conditions achieved in a 2000-L bioreactor cellculture process run.

Previous experiments demonstrated that a shake flask microcarrierbatch-refeed cell culture process successfully replicates the cellgrowth and productivity achieved in a larger 2000-L bioreactor cellculture process run (U.S. Provisional Patent Application No.61/768,085). Described in this Example is a shake tube microcarrierbatch re-feed cell culture process that accurately simulates therecombinant human a-galactosidase 2000-L bioreactor perfusion cellculture process.

Materials and Equipment

Fabrazyme Cell Source

The human recombinant a-galactosidase-producing cells used in each cellculture process run were derived from the same cell bank, in order toensure comparability between each cell culture process run. The cellsare stably transformed with a nucleic acid that encodes a secreted formof human recombinant α-galactosidase. A growth medium (925 medium with10% Dulbecco's bovine serum, pH 7.3, and 0.1% Pluronic F-68) was usedduring the cell bank culture expansion process.

Equipment

The following equipment was used to perform the experiments described inthis Example: a Multitron Shaker Incubator (Appropriate TechnicalResources, Inc., Model No. AG CH-4103), a Beckman Coulter Vi-Cell CellViability Analyzer (Beckman Coulter, Inc., Model XR), a YSI BiochemistryAnalyzer (Yellow Springs Instruments, Inc. Model No. 2700 Select), and aBlood Gas Analyzer (Bayer AG, Model No. 248).

Experimental Design

The inoculum used for the exemplary shake tube microcarrier batchre-feed cell culture process runs was generated from a seed cultureexpansion of a thawed vial of recombinant humana-galactosidase-producing CHO cells. After five days of expansion of thethawed cells in 925 medium with 10% DBS, pH 7.3, and 0.1% Pluronic F-68,the seed culture was used to inoculate a shake tube (at a finalconcentration of 0.25×10⁶ viable cells/mL in the shake tube) containinga sterilized microcarrier slurry (Cytopore II, final concentration of1.5 g/L in the growth medium) and growth medium (925 medium with 6% DBS,pH 7.0, and 0.1% Pluronic F-68), which initiates the growth phase of thecell culture. The working volume for the shake tubes (50 mL totalvolume) was designed to be 7 mL. The cultures were maintained at 37° C.or 36° C., 80% relative humidity, and 5% CO₂. Three differentfrequencies of rotary agitation were tested for their capability tosupport and maintain cell growth and productivity: 120 RPM, 140 RPM, and160 RPM. When each culture reached a target cell density of between2.0×10⁶ to 3.0×10⁶ viable cells/mL, the transition phase was initiatedby changing the liquid culture medium to a different production liquidculture medium (925 medium, pH 6.85-7.05, and 0.1% Pluronic F-68) andshifting the temperature to 32° C. After 5 days of the transition phase,the temperature was shifted back to 37° C. or 36° C., and the cultureswere maintained with production liquid culture medium. Media exchangewas initiated on the third day of the growth phase and was continueduntil the end of each cell culture process run, with a daily batchre-feed exchange of 70% of the initial volume of the liquid culturemedium present in the shake tube at the start of the cell cultureprocess run. On each day, starting on the third day of the growth phase,media exchange was performed by briefly stopping the agitation of theshake tube, placing the shake tube upright, allowing the microcarriersto settle to the bottom of the shake tube for about 1 minute to about 2minutes, and removing from the shake tube a volume of the liquid culturemedium that is 70% of the initial volume of the liquid culture mediumpresent in the shake tube at the start of the culture when the culturemedium is free of microcarriers by visual inspection, and then shortlythereafter, adding a volume of liquid culture medium that issubstantially the same volume as the volume of liquid culture mediumremoved.

The inoculum used for the control shake flask microcarrier cell cultureprocess runs was generated from a seed culture expansion of a thawedvial of the same recombinant human α-galactosidase-producing CHO cellsused to inoculate the shake tube microcarrier cell culture process runsdescribed in this Example. After five days of expansion of the thawedcells in 925 medium with 10% DBS, pH 7.3, and 0.1% Pluronic F-68, theseed culture was used to inoculate a shake flask (at a finalconcentration of 0.25×10⁶ viable cells/mL in the shake flask) containinga sterilized microcarrier slurry (CytoPore2, GE Healthcare, Piscataway,N.J.; final concentration of 1.5 g/L; average size 200-280 μm; averagepore size 30 μm) and growth medium (925 medium with 6% DBS, pH 7.0, and0.1% Pluronic F-68), which initiates the growth phase of the cellculture. The cultures were maintained at 37° C. or 36° C., 95 RPM, 80%relative humidity, and 5% CO₂. When the culture reached a target celldensity of between 2.5×10⁶ to 3.0×10⁶ viable cells/mL, the transitionphase was initiated by changing the liquid culture medium to a differentproduction liquid culture medium (925 medium, pH 6.85-7.05, and 0.1%Pluronic F-68) and shifting the temperature to 32° C. After 5 days oftransition phase, the temperature was shifted back to 37° C. or 36° C.,and the cultures were maintained with production liquid culture medium.Medium exchange was initiated on the third day of the growth phase andwas continued until the end of culture, with a daily batch re-feedexchange of 70% of the initial volume of the liquid culture mediumpresent in the shake flask at the start of the culture. On each day,starting on the third day of the growth phase, medium exchange wasperformed by briefly stopping the agitation of the shake flask, allowingthe microcarriers to settle to the bottom of the shake flask in abiosafety hood. In some instances, the shake flask was placed in a rackwhich positions the shake flask at a 45 degree angle with respect to thehorizon or the benchtop while the microcarriers settled to the bottom ofthe shake flask in order to improve medium exchange. After themicrocarriers have settled to the bottom of the shake flask, a volume ofliquid culture medium that is 70% of the initial volume of the liquidculture medium present in the shake flask is removed from the shakeflask, and then shortly thereafter, a volume of liquid culture mediumthat is substantially the same volume as the volume of liquid culturemedium removed is added to the shake flask.

A summary of the process conditions and sampling schedule for each shaketube cell culture process run and shake flask cell culture process runis provided in Table 1.

On pre-determined culture days, the following culture parameters wereanalyzed in each cell culture process run: viable and suspension celldensity, pCO2, pO2, pH, and glucose, lactate, glutamine, and glutamateconcentration. Viable cell concentration is a critical parameter duringthe growth and transition phase in each cell culture process run, as theinitiation of the transition phase is based on cell concentration.However, in order to reduce cell loss, the frequency of sampling used todetermine viable cell density during the harvest phase for the shaketube cell culture process runs was dramatically reduced. Other culturecharacteristics, such as metabolic profiles of the spent media andproductivity can be relied upon to monitor the culture performance ineach cell culture process run. The consumption rates of glucose andglutamine, as well as production rates

TABLE 1 Summary of Cell Culture Process Run Conditions and SamplingSchedule. Shake Flasks Shake Tubes Process Working Volume 60 mL 7 mLCondition Vessel Volume 250 mL 50 mL Incubator Shaking 95 RPM 120, 140,160 RPM Speed Sampling Cell Count 3 samples/week 3 samples/week forSchedule G/T phase; 1 sample/week for H phase Metabolites 3 samples/week3 samples/week BGA (pH, pCO₂, pO₂) 3 samples/week 3 samples/weekProductivity 3 samples/week 3 samples/weekof lactate and glutamate in each cell culture process run werecalculated using Equations 1-4 (below). Titer samples were collected andstored at −20° C. until the assay was performed to measure therecombinant human α-galactosidase activity. Cumulative volumetricproductivity was calculated using Equation 5 (below). One full cellculture process run was performed with a triplicate set of shake tubesand shake flasks (conditions described below) for each condition. Theaverage and standard deviation of the data were calculated, and areshown in each of FIGS. 1-9.

Gluc_(cons)=([Gluc]_(m) −[Gluc] _(c))*PR   Equation 1

Lac_(prod)=[Lac]_(c) *PR   Equation 2

Gln _(cons)=([Gln] _(m) −[Gln] _(c))*PR   Equation 3

Glu_(cons)=([Glu]_(c)−[Glu]_(m))*PR   Equation 4

ΣVPR=Σ(titer*PR)   Equation 5

Gluc_(m): Glucose concentration in fresh media (4.5 g/L)

Gluc_(c): Glucose concentration in spent media (g/L)

Lac_(c): Lactate concentration in spent media (g/L)

Gln_(m): Glutamine concentration in fresh media (4 mM)

Gln_(c): Glutamine concentration in spent media (mM)

Glu_(m): Glutamate concentration in fresh media (0.8 mM)

Glu_(c): Glutamate concentration in spent media (mM)

PR: Perfusion rate per day (re-feed rate)

Titer: rhαa-Gal activity (U/L)

VPR: Volumetric productivity (U/L/d)

Results

Growth, Transition, and Early Harvest Phase

Culture performance during the growth phase through transition phase isimportant to a successful cell culture process run. Table 2 provides asummary of growth/transition parameters of all the cell culture processruns tested. Shake tube cell culture process runs performed using ashaking speed of 140 RPM reached the target transition cell density of2.86×10⁶ cells/mL on the seventh day of the growth phase. Using thisspecific throw (orbit) diameter and shake tube, the rotary agitationrate of 160 RPM did not support cell growth and this cell cultureprocess run was terminated in early growth phase. This may be due to thehigh shear stress the culture was experiencing using this specificcombination of shaking speed, throw (orbit) diameter, and shake tube.The shake tube cell culture process runs performed using an agitationfrequency of 120 RPM were forced into the transition phase on the ninthday of the growth phase, with a transition cell density of 1.12×10⁶cells/mL (a density that is outside of the exemplary target transitioncell density range of 2-3×10⁶ cells/mL). Visual inspection of the cellculture process runs performed at a frequency of agitation of 120 RPMrevealed clumping of the microcarrier cell aggregates, mostly likely dueto the inadequate shaking speed.

The viable cell density (Xv) profiles for each cell culture process runis shown in FIG. 1. Due to the low transition density for the shake tubecell culture process runs performed using an agitation frequency of 120RPM, the viable cell density in these cell culture process runs was notable to recover from the serum washout and decrease in culturetemperature that occurs during the transition phase. These cell cultureprocess runs were terminated on harvest day 12. The shake tube cellculture process runs performed using an agitation frequency of 140 RPM(FIG. 1) were able to successfully complete the transition phase andmaintain a steady viable cell density profile similar to that of thecontrol shake flask cell culture process runs through transition phaseand early harvest phase.

TABLE 2 Summary of Growth Phase Duration and Transition Density of theCell Culture Process Runs Experimental Condition Transition DensityGrowth Day to Transition Shake Tube 120 RPM 1.12 E6 cells/mL G9 ShakeTube 140 RPM 2.86 E6 cells/mL G7 Shake Tube 160 RPM Terminated due tolow cell growth Shake Flasks 2.48 E6 cells/mL G9

Culture Growth

The growth and performance of each cell culture process run wasmonitored. Culture growth for the shake tube cell culture process runsperformed using an agitation frequency of 140 RPM, as represented byviable cell concentration (FIG. 1), showed a consistent increase in cellconcentration through growth phase and early transition phase to reach amaximum of approximately 2.5-3×10⁶ viable cells/mL. As these cultureswere adapting to serum-free medium (beginning in transition phase andthrough harvest phase), there was a dramatic decrease in viable cellconcentration during the early harvest phase. The cultures thenstabilized between 0.5-1×10⁶ viable cells/mL throughout the harvestphase of the cell culture process runs. The growth profiles of the shaketube cell culture process runs performed using an agitation frequency of140 RPM and the shake flask cell culture process runs were comparablethroughout the culture period.

Culture Productivity

Culture productivity was monitored throughout the culture period tocompare the performance of the shake tube cell culture process runsperformed using an agitation frequency of 140 RPM and the shake flaskcell culture process runs. The culture productivity for both the shaketube cell culture process runs and the shake flask cell culture processruns peaked at late transition phase/early harvest phase. However, asthe cultures were adapting to serum-free medium, a sharp decline inproductivity was observed. The cultures recovered from this troughperiod in early harvest phase, and stabilized through the end of cellculture process runs. When comparing the productivity profile of theshake tube cell culture process runs performed at an agitation frequencyof 140 RPM to the shake flask cell culture process runs, a differencewas observed during the mid-harvest phase. The shake tube cell cultureprocess runs performed using an agitation frequency of 140 RPM were ableto produce a second productivity peak during the mid-harvest phase (H15to H35). This second peak of productivity was also reflected in thecumulative volumetric productivity profile for these cultures (see, FIG.3). Historical results from previous shake flask cell culture processruns show that productivity was maintained at a steady level (600 U/L)from mid-harvest phase to end of each cell culture process run. Anincrease in viable cell concentration was not observed for the shaketube cell culture process runs performed using an agitation frequency of140 RPM during the mid-harvest phase, which indicated that theproductivity peak was a result of an increase in specific cellproductivity.

Culture Metabolism

Cellular metabolism was monitored in each cell culture process runthrough the measurement of the glucose, lactate, glutamine, andglutamate concentrations in the spent media. The glucose consumptionrate (FIG. 4) and lactate production rate (FIG. 5) were calculated fromthe glucose and lactate concentration present in the spent media andfeed media samples. Overall, both glucose consumption and lactateproduction corroborated with the cell growth profile in each cellculture process run (FIG. 1). The most dynamic periods of the cultureoccurred at two different culture stages: i) where cell proliferationoccurred in the growth phase (with the serum-containing medium), and ii)mid- to late-harvest phase, when a re-growth period occurred, and anincrease in both viable cell density and metabolic activity wereobserved. Slightly higher glucose consumption rate and lactateproduction rate were observed in the shake tube cell culture processruns performed using an agitation frequency of 140 RPM when compared tothe shake flask cell culture process runs during mid-harvest phase(FIGS. 4 and 5). This was also when the shake tube cell culture processruns performed using an agitation frequency of 140 RPM showed a higherproductivity than the shake flask cell culture process runs. Comparabletrends for glutamine consumption rate (FIG. 6) and glutamate productionrate were observed for the shake tube shake tube cell culture processruns performed using an agitation frequency of 140 RPM and the shakeflask cell culture process runs.

Culture pH, pCO₂, and pO₂

Culture pH, pCO₂, and pO₂ profiles were monitored in each cell cultureprocess run using a blood gas analyzer (BGA) during sampling. The shaketube cell culture process runs performed using an agitation frequency of140 RPM and the shake flasks cell culture process runs had comparablepH, pCO₂, and pO₂ profiles (FIGS. 7-9) over most of the duration of theprocess runs. The pH profiles (FIG. 7) corroborated with the viable cellconcentration dip that occurs in both the shake tube cell cultureprocess runs performed using an agitation frequency of 140 RPM and theshake flask cell culture process runs during the transition period. ThepCO₂ profiles (FIG. 9) show that a level of pCO₂ between 30-40 mmHg wasmaintained in each cell culture process run, which correlates to the 5%CO₂ setting of the incubator.

In sum, the exemplary batch re-feed shake tube culture model describedin this Example was demonstrated to be an acceptable model for thelarge-scale production of recombinant human alpha-galactosidase using a2000-L bioreactor. The exemplary shake tube cell culture process rundescribed in the Example demonstrated similar cell growth andproductivity to that of a shake flask cell culture process run that waspreviously demonstrated to be an acceptable model for the large-scaleproduction of recombinant human alpha-galactosidase using a 2000-Lbioreactor.

Under the present experimental conditions (e.g., use of a specific throw(orbit) diameter), agitation of the shake tube cultures at a frequencyof 140 RPM was shown to achieve a target transition cell density of2-3×10⁶ cells/mL (a cell density of 2.86×10⁶ cells/mL achieved on theseventh day of the growth phase), and to maintain a growth profilesimilar to that achieved using the control shake flask cell cultureprocess runs. Unlike the shake flask cell culture process runs, theshake tube cell culture process runs performed using an agitationfrequency of 140 RPM were able to produce a second productivity peakduring the mid-harvest phase. As an increase in viable cell density wasnot observed during this period in the shake tube cell culture processruns, the increase in productivity is attributed to an increase inspecific cell productivity. The changes in the culture metabolism areconsistent with these findings—with both the glucose consumption andlactate production during the mid-harvest phase measured to be higher inthe shake tube cell culture process runs performed using an agitationfrequency of 140 RPM than in the shake flask cell culture process runs.Other metrics, such as culture pH, pCO₂, and pO₂, were found to besimilar between the shake tube cell culture process runs performed.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-110. (canceled)
 111. A method for testing a manufacturing process formaking a recombinant protein, the method comprising: providing a shaketube containing an adherent Chinese hamster ovary (CHO) cell containinga nucleic acid encoding a recombinant protein disposed in a first liquidculture medium, wherein the first liquid culture medium occupies about10% to about 30% of the volume of the shake tube and contains aplurality of microcarriers at a concentration of about 1.0 g/L to about15.0 g/L; incubating the shake tube for a period of time at about 32° C.to about 39° C. and with a rotary agitation of about 130 revolutions perminute (RPM) to about 150 RPM; after about the first 48 to 96 hours ofthe period of time, continuously or periodically removing a first volumeof the first liquid culture medium and adding to the first liquidculture medium a second volume of a second liquid culture medium,wherein the first and second volumes are about equal, and the methodachieves a viable cell density of greater than 2.0×10⁶ cells/mL in thefirst liquid culture medium or a combination of the first and secondliquid culture medium at some point during the period of time; detectingthe recombinant protein in the cell or in the first and/or secondculture medium; and comparing the amount of recombinant protein presentin the adherent CHO cell or in the first and/or second culture medium toa reference level of recombinant protein.
 112. The method of claim 111,wherein the reference level of recombinant protein is a level ofrecombinant protein produced using a different culturing method. 113.The method of claim 112, wherein the different culturing method utilizesa different first or second liquid culture medium, a different adherentCHO cell, a different temperature, a different level of agitation, adifferent shake tube, or a different microcarrier.
 114. The method ofclaim 112, wherein the different culturing method utilizes a differentraw material, anti-clumping agent, or chemically-defined liquid culturemedium.
 115. The method of claim 111, wherein the method is used toperform high throughput cell culture experiments to perform adesign-of-experiment (DOE) or a quality-by- design (QBD) study.
 116. Themethod of claim 111, wherein the shake tube has a volume of betweenabout 10 mL to about 100 mL.
 117. The method of claim 111, wherein theadherent CHO cell is suspended in about 2 mL to about 20 mL of the firstliquid culture medium.
 118. The method of claim 111, wherein therecombinant protein is a secreted immunoglobulin, a secreted enzyme, asecreted growth factor, a secreted protein fragment, or a secretedengineered protein, and wherein the recombinant protein is recoveredfrom the first or second culture medium.
 119. The method of claim 111,wherein the recombinant protein is recovered from the adherent CHO cell.120. The method of claim 111, wherein the recombinant protein is animmunoglobulin, an enzyme, a growth factor, a protein fragment, or anengineered protein.
 121. The method of claim 111, wherein the removingof the first volume of the first liquid culture medium and the adding ofthe second volume of the second liquid culture medium is performedsimultaneously.
 122. The method of claim 111, wherein the removing ofthe first volume of the first liquid culture medium and the adding ofthe second volume of the second liquid culture medium is performedcontinuously.
 123. The method of claim 111, wherein the removing of thefirst volume of the first liquid culture medium and the adding of thesecond volume of the second liquid culture medium is performedperiodically.
 124. The method of claim 111, wherein the first volume ofthe first liquid culture medium removed and the second volume of thesecond liquid culture medium added are increased over time.
 125. Themethod of claim 111, wherein the first liquid culture medium and/orsecond liquid culture medium is selected from the group consisting of: achemically-defined liquid culture medium, a serum-free liquid culturemedium, a serum-containing liquid culture medium, an animal-derivedcomponent free liquid culture medium, and a protein-free medium. 126.The method of claim 111, wherein the shake tube is incubated at areactor angle of about 25 degrees to about 90 degrees from horizontal.127. A method of optimizing a manufacturing process of producing arecombinant protein, the method comprising: providing a shake tubecontaining an adherent Chinese hamster ovary (CHO) cell containing anucleic acid encoding the recombinant protein disposed in a first liquidculture medium, wherein the first liquid culture medium occupies about10% to about 30% of the volume of the shake tube and contains aplurality of microcarriers at a concentration of about 1.0 g/L to about15.0 g/L; incubating the shake tube for a period of time at about 32° C.to about 39° C. and with a rotary agitation of about 130 revolutions perminute (RPM) to about 150 RPM; and after about the first 48 to 96 hoursof the period of time, continuously or periodically removing a firstvolume of the first liquid culture medium and adding to the first liquidculture medium a second volume of a second liquid culture medium,wherein the first and second volumes are about equal, and the methodachieves a viable cell density of greater than 2.0×10⁶ cells/mL in thefirst liquid culture medium or a combination of the first and secondliquid culture medium at some point during the period of time; detectingthe recombinant protein in the adherent CHO cell or in the first and/orsecond culture medium; comparing the amount of recombinant proteinpresent in the adherent CHO cell or in the first and/or second culturemedium to a reference level of recombinant protein produced by adifferent method; and identifying and removing or altering in amanufacturing process any culture components or parameters that areassociated with a decrease in the amount of recombinant protein producedas compared to the reference level, or identifying and adding to amanufacturing process any culture components or parameters that areassociated with an increase in the amount of recombinant proteinproduced as compared to the reference level.
 128. The method of claim127, wherein the shake tube has a volume of between about 10 mL to about100 mL.
 129. The method of claim 127, wherein the adherent CHO cell issuspended in about 2 mL to about 20 mL of the first liquid culturemedium.
 130. The method of claim 127, wherein the shake tube isincubated at a reactor angle of about 25 degrees to about 90 degreesfrom horizontal.